Mutated anti-TNFα antibodies and methods of their use

ABSTRACT

The present invention is directed to modified antibodies, including anti-TNFα antibodies, in which C-terminal amino acids of heavy chain sequences are modified from a native sequence of proline-glycine-lysine (“PGK”) to one that includes a proline positioned between the glycine and lysine, resulting in a C-terminal sequence of proline-glycine-proline-lycine (“PGPK”). The invention further provides methods of producing and using such antibodies.

RELATED APPLICATION INFORMATION

This application claims priority to U.S. Provisional Application No.61/784,430, filed on Mar. 14, 2013, and U.S. Provisional Application No.61/892,710, filed on Oct. 18, 2013, the entire contents of each of whichare expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

Large-scale production of antibodies for biopharmaceutical applicationsinvolves the use of cell cultures that are known to produce antibodies,and antigen-binding portions thereof, exhibiting varying levels ofheterogeneity. This heterogeneity may comprise isoforms of the antibodywherein the difference between the isoforms is dependent on charge. Someof these charge variants (or isoforms) may lead to decreased productefficacy and stability. Alternatively, adding a basic overall charge toan antibody may contribute positive attributes to the antibody such asincreased tissue penetration.

One source of antibody heterogeneity involves C-terminal lysineresidues, such as those typically found on the heavy chains of antibodymolecules, which can be lost during both the purification process and/orstorage of the final composition, resulting in compositions comprisingantibody species that can vary at their C-terminus as to whether alysine residue is present. For example, C-terminal lysines canpotentially be present on both the heavy chains of an antibody (Lys 2),on either one of the heavy chains (Lys 1), or neither of them (Lys 0).Since lysine carries a positive charge, antibodies lacking the basicC-terminal lysine(s) differ in their charge state from ones that containthe lysine, so that the distribution of lysine variants (% Lys 0, % Lys1, % Lys 2 of the total Lysine Sum) can be detected by ion-exchangechromatographic methods, for example, using a ProPac WCX-10 WeakCation-Exchange column for the high-resolution separation of proteinisoforms (Dionex, Calif.), and subsequently quantified.

The development of compositions comprising antibodies, orantigen-binding portions thereof, with either higher or lower levels oflysine variants to increase efficacy and stability of antibody productsis an important, to date unmet, need in the biopharmaceutical industry.

SUMMARY OF THE INVENTION

The present invention provides antibodies, and antigen-binding portionsthereof, comprising mutations in their C-terminal residues that decreaseC-terminal processing of lysine residues, thereby leading to moreefficacious and stable antibody compositions having one or more terminallysine residues. The present invention also provides compositionscomprising antibodies, and antigen-binding portions thereof, withdecreased levels of C-terminal processing of lysines.

Accordingly, in one aspect, the invention provides an antibody, e.g., anIgG₁ antibody, or antigen-binding portion thereof, comprising aC-terminal heavy chain sequence of proline-glycine-proline-lysine (PGPK)(SEQ ID NO:9). In one embodiment, the antibody, or antigen-bindingportion thereof, is a human antibody, or antigen-binding portionthereof. In one embodiment, the antibody, or antigen-binding portionthereof, is an anti-TNFα antibody, or antigen-binding portion thereof.In another embodiment, the antibody, or antigen-binding portion thereof,comprises a light chain variable region (LCVR) having a CDR1 domaincomprising the amino acid sequence of SEQ ID NO:7, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO:5, and a CDR3 domaincomprising the amino acid sequence of SEQ ID NO:3; and a heavy chainvariable region (HCVR) having a CDR1 domain comprising the amino acidsequence of SEQ ID NO:8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO:6, and a CDR3 domain comprising the amino acidsequence of SEQ ID NO:4. In another embodiment, the antibody, orantigen-binding portion thereof, comprises a LCVR comprising the aminoacid sequence set forth in SEQ ID NO:1 and a HCVR comprising the aminoacid sequence set forth in SEQ ID NO:2. In one embodiment, the antibody,or antigen-binding portion thereof, comprises adalimumab, or anantigen-binding portion thereof. In another embodiment, the antibody, orantigen-binding portion thereof, is an IgG₁, an IgG₂, an IgG₃, or anIgG₄ antibody, or antigen-binding portion thereof. In one embodiment,the antibody, or antigen-binding portion thereof, is an IgG₁ antibody,or antigen-binding portion thereof.

In one embodiment, the antibody, e.g., an IgG₁ antibody, orantigen-binding portion thereof, is resistant to C-terminal processingby a carboxypeptidase. In one embodiment, the antibody, orantigen-binding portion thereof, exhibits no C-terminal processing by acarboxypeptidase. In one embodiment, the carboxypeptidase iscarboxypeptidase B. In another embodiment, the carboxypeptidase iscarboxypeptidase U.

In one embodiment, the antibody, e.g., an IgG₁ antibody, orantigen-binding portion thereof, exhibits increased tissue, e.g.,cartilage, penetration as compared to an antibody, or antigen-bindingportion thereof, comprising a C-terminal heavy chain sequence ofproline-glycine-lysine (PGK) (SEQ ID NO:10).

In one embodiment, the antibody, e.g., an IgG₁ antibody, orantigen-binding portion thereof, exhibits increased TNFα affinity ascompared to an antibody, or antigen-binding portion thereof, comprisinga C-terminal heavy chain sequence of proline-glycine-lysine (PGK) (SEQID NO:10).

In another embodiment, the antibody, e.g., an IgG₁ antibody, orantigen-binding portion thereof, exhibits reduced tissue, e.g.,cartilage, destruction as compared to an antibody, or antigen-bindingportion thereof, comprising a C-terminal heavy chain sequence ofproline-glycine-lysine (PGK) (SEQ ID NO:10).

In another embodiment, the antibody, e.g., an IgG₁ antibody, orantigen-binding portion thereof, exhibits reduced bone erosion, reducedsynovial proliferation, reduced cell infiltration, reduced chondrocytedeath, or reduced proteoglycan loss as compared to an antibody, orantigen-binding portion thereof, comprising a C-terminal heavy chainsequence of proline-glycine-lysine (PGK) (SEQ ID NO:10).

In another embodiment, the antibody, e.g., an IgG₁ antibody, orantigen-binding portion thereof, exhibits increased protection againstthe development of arthritic scores or increased protection against thedevelopment of histopathology scores as compared to an antibody, orantigen-binding portion thereof, comprising a C-terminal heavy chainsequence of proline-glycine-lysine (PGK) (SEQ ID NO:10) whenadministered to an animal model of arthritis.

In another embodiment, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(d) of about 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ s⁻¹ or less.

In another aspect, the invention provides a composition comprising anantibody, e.g., an IgG₁ antibody, or antigen-binding portion thereof,wherein the composition comprises less than about 50% lysine variantspecies that lack a C-terminal lysine (Lys 0). In one embodiment, thecomposition comprises less than about 25% lysine variant species thathave one C-terminal lysine (Lys 1). In another embodiment, thecomposition comprises at least about 70% lysine variant species thathave two C-terminal lysines (Lys 2). In another embodiment, thecomposition comprises at least about 80% lysine variant species thathave two C-terminal lysines (Lys 2). In another embodiment, thecomposition comprises at least about 90% lysine variant species thathave two C-terminal lysines (Lys 2). In another embodiment, thecomposition comprises at least about 95% lysine variant species thathave two C-terminal lysines (Lys 2).

In one embodiment, the composition comprises less than about 10% acidicspecies, wherein the acidic species comprise a first acidic speciesregion (AR1) and a second acidic species region (AR2). In oneembodiment, the composition comprises about 3% acidic species. Inanother embodiment, the composition comprises less than about 1% AR1. Inanother embodiment, the composition comprises about 0% AR1. In anotherembodiment, the composition comprises less than about 4% AR2. In anotherembodiment, the composition comprises about 3% AR2. In anotherembodiment, the composition comprises about 0% AR1 and about 3% AR2.

In another aspect, the invention provides a composition comprising anantibody, or antigen-binding portion thereof, wherein the compositioncomprises at least about 70% lysine variant species that have twoC-terminal lysines (Lys 2). In one embodiment, the composition comprisesat least about 75% lysine variant species that have two C-terminallysines (Lys 2). In another embodiment, the composition comprises atleast about 80% lysine variant species that have two C-terminal lysines(Lys 2). In another embodiment, the composition comprises at least about85% lysine variant species that have two C-terminal lysines (Lys 2). Inanother embodiment, the composition comprises at least about 90% lysinevariant species that have two C-terminal lysines (Lys 2). In anotherembodiment, the composition comprises at least about 100% lysine variantspecies that have two C-terminal lysines (Lys 2).

In another embodiment, the composition comprises less than about 10%acidic species, wherein the acidic species comprise a first acidicspecies region (AR1) and a second acidic species region (AR2). In oneembodiment, the composition comprises about 3% acidic species. Inanother embodiment, the composition comprises less than about 1% AR1. Inone embodiment, the composition comprises about 0% AR1. In anotherembodiment, the composition comprises less than about 4% AR2. In anotherembodiment, the composition comprises about 3% AR2. In one embodiment,the composition comprises about 0% AR1 and about 3% AR2.

In one embodiment, the antibody, or antigen-binding portion thereof, isan anti-TNFα antibody, or antigen-binding portion thereof. In oneembodiment, the antibody, or antigen-binding portion thereof, comprisesa light chain variable region (LCVR) having a CDR1 domain comprising theamino acid sequence of SEQ ID NO:7, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO:5, and a CDR3 domain comprising the aminoacid sequence of SEQ ID NO:3; and a heavy chain variable region (HCVR)having a CDR1 domain comprising the amino acid sequence of SEQ ID NO:8,a CDR2 domain comprising the amino acid sequence of SEQ ID NO:6, and aCDR3 domain comprising the amino acid sequence of SEQ ID NO:4. In oneembodiment, the antibody, or antigen-binding portion thereof, comprisesa LCVR comprising the amino acid sequence set forth in SEQ ID NO:1 and aHCVR comprising the amino acid sequence set forth in SEQ ID NO:2. Inanother embodiment, the antibody, or antigen-binding portion thereof,comprises adalimumab, or an antigen-binding portion thereof.

In one embodiment, the antibody, or antigen-binding portion thereof, isresistant to C-terminal processing by a carboxypeptidase. In oneembodiment, the antibody, or antigen-binding portion thereof, exhibitsno C-terminal processing by a carboxypeptidase. In one embodiment, thecarboxypeptidase is carboxypeptidase B. In another embodiment, thecarboxypeptidase is carboxypeptidase U.

In one embodiment, the antibody, e.g., an IgG₁ antibody, orantigen-binding portion thereof, exhibits increased cartilage tissuepenetration, increased TNFα affinity, reduced cartilage destruction,reduced bone erosion, or reduced synovial proliferation as compared toan antibody, or antigen-binding portion thereof, comprising a C-terminalheavy chain sequence of proline-glycine-lysine (PGK) (SEQ ID NO:10).

In another embodiment, the antibody, e.g., an IgG₁ antibody, orantigen-binding portion thereof, exhibits reduced cell infiltration,reduced chondrocyte death, or reduced proteoglycan loss as compared toan antibody, or antigen-binding portion thereof, comprising a C-terminalheavy chain sequence of proline-glycine-lysine (PGK) (SEQ ID NO:10).

In another embodiment, the antibody, or antigen-binding portion thereof,exhibits increased protection against the development of arthriticscores as compared to an antibody, or antigen-binding portion thereof,comprising a C-terminal heavy chain sequence of proline-glycine-lysine(PGK) (SEQ ID NO:10) when administered to an animal model of arthritis.

In one embodiment, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(d) of about 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ s⁻¹ or less.

In another aspect, the invention provides a nucleic acid moleculeencoding an antibody, or antigen-binding portion thereof, of theinvention. In another aspect, the invention provides a vector comprisingthe nucleic acid molecule encoding an antibody, or antigen-bindingportion thereof, of the invention. In another aspect, the inventionprovides a host cell comprising the vector.

In another aspect, the invention provides a composition comprising anantibody, or antigen-binding portion thereof, of the invention, and apharmaceutically acceptable carrier.

In another aspect, the invention provides a kit comprising an antibody,or antigen-binding portion thereof, of the invention, and instructionsfor use.

In another aspect, the invention provides a method of treating a subjecthaving a disorder in which TNFα activity is detrimental, the methodcomprising administering a therapeutically effective amount of anantibody, or antigen-binding portion thereof, of the invention, or acomposition of the invention to the subject, thereby treating theTNFα-associated disease or disorder. In one embodiment, the disorder inwhich TNFα activity is detrimental is selected from the group consistingof rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis,Crohn's Disease and plaque psoriasis. In one embodiment, the disorder inwhich TNFα activity is detrimental is selected from the group consistingof active axial spondyloarthritis and non-radiographic axialspondyloarthritis.

In another aspect, the invention provides a method of modifying anantibody, or antigen-binding portion thereof, to make it resistant toC-terminal processing by a carboxypeptidase, the method comprisingmodifying the three C-terminal amino acids of the heavy chain sequence,proline-glycine-lysine (“PGK”) (SEQ ID NO:10), of the antibody, orantigen-binding portion thereof, to introduce a proline between glycineand lysine, thereby producing an antibody, or antigen-binding portionthereof, comprising a C-terminal heavy chain sequence ofproline-glycine-proline-lysine (“PGPK”) (SEQ ID NO:9) which is resistantto C-terminal processing by a carboxypeptidase.

In another aspect, the invention provides a method of increasing thecartilage tissue penetration of an antibody, or antigen-binding portionthereof, the method comprising modifying the three C-terminal aminoacids of the heavy chain sequence, proline-glycine-lysine (SEQ IDNO:10), of the antibody, or antigen-binding portion thereof, tointroduce a proline between glycine and lysine, thereby producing anantibody, or antigen-binding portion thereof, comprising a C-terminalheavy chain sequence of proline-glycine-proline-lysine (“PGPK”) (SEQ IDNO:9) which exhibits increased cartilage tissue penetration.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a WCX-10 chromatogram of a sample of Adalimumabindicating the presence of particular charge variants including Lys 0,Lys 1, and Lys 2.

FIG. 2 depicts a subsequent analysis of the WCX-purified materialindicating that the individual charge variants are resolvable from oneanother.

FIG. 3 depicts the results of incubation of Adalimumab with or withoutcarboxypeptidase B. In the presence of carboxypeptidase B the C-terminallysines are cleaved leaving only Lys 0 species (with and without aparticular glycol-modification (Gal)).

FIG. 4 depicts the results of incubating a modified anti-TNFα antibodywith and without carboxypeptidase B. Both the treated and untreatedsamples display a homogenous population with the presence of anunprocessed GPK c-terminus.

FIG. 5 depicts the results of incubation of a modified anti-TNFαantibody in three different plasma samples. The modified anti-TNFαantibody retains both C-terminal lysines in all conditions.

FIG. 6 depicts the results of incubation of Adalimumab in the threedifferent plasma samples. The antibody only retains C-terminal lysineresidues in human plasma with citrate used as an anticoagulent. Withoutbeing bound by theory, the citrate presumably chelates the active sitemetal cation and inhibits the carboxypeptidase U activity.

FIG. 7 depicts intact LC/MS analysis of a modified anti-TNFα antibodyobtained from terminal mouse bleed of an animal dosed at 5 mg/Kg. Therecovered Mab has an intact GPK motif on both heavy chains.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides antibodies, and antigen-binding portionsthereof, comprising mutations in their C-terminal residues that decreaseC-terminal processing of lysine residues, thereby leading to moreefficacious and stable antibody compositions. The present invention alsoprovides compositions comprising antibodies, and antigen-bindingportions thereof, with decreased levels of C-terminal processing oflysines.

The present invention is based, at least in part, on the discovery thatthe C-terminal lysines of antibodies in pharmaceutical compositions canbe lost during both the purification process and/or storage of the finalcomposition, resulting in compositions comprising individual antibodyspecies that can vary at their C-terminus as to whether a lysine residueis present. Moreover, terminal lysines can be cleaved by enzymes, suchas a carboxypeptidase, in vivo. The inventors of the instant applicationhave surprisingly discovered that modifying an antibody, orantigen-binding portion thereof, comprising a C-terminal heavy chainsequence of proline-glycine-lysine (“PGK”) by inserting a prolinebetween the glycine and the lysine (proline-glycine-proline-lysine, or“PGPK”) prevents the C-terminal processing of the antibody heavy chainand leads to more efficacious and stable antibody compositions.

In order that the present invention may be more readily understood,certain terms are first defined.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivative thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art and non-limitingembodiments of which are discussed herein.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA1 andIgA2) or subclass. The present invention is particularly useful for IgG₁antibodies.

As used herein, the term “adalimumab”, also known by its trade nameHUMIRA® (AbbVie) refers to a human IgG₁ antibody that binds human tumornecrosis factor α (TNFα). In general, the heavy chain constant domain 2(CH2) of the adalimumab IgG-Fc region is glycosylated through covalentattachment of oligosaccharide at asparagine 297 (Asn-297). The lightchain variable region of adalimumab is provided herein as SEQ ID NO:1,and the heavy chain variable region of adalimumab is provided herein asSEQ ID NO:2. Adalimumab comprises a light chain variable regioncomprising a CDR1 of SEQ ID NO:7, a CDR2 of SEQ ID NO:5, and a CDR3 ofSEQ ID NO:3. Adalimumab comprises a heavy chain variable regioncomprising a CDR1 of SEQ ID NO:8, a CDR2 of SEQ ID NO:6 and CDR3 of SEQID NO:4. The light chain of adalimumab is provided herein as SEQ IDNO:13, and the heavy chain of adalimumab is provided herein as SEQ IDNO:14. Adalimumab is described in U.S. Pat. Nos. 6,090,382; 6,258,562;6,509,015; 7,223,394; 7,541,031; 7,588,761; 7,863,426; 7,919,264;8,197,813; 8,206,714; 8,216,583; 8,420,081; 8,092,998; 8,093,045;8,187,836; 8,372,400; 8,034,906; 8,436,149; 8,231,876; 8,414,894;8,372,401, and PCT Publication No. WO2012/065072, the entire contents ofeach which are expressly incorporated herein by reference in theirentireties. Adalimumab is also described in “Highlights of PrescribingInformation” for HUMIRA® (adalimumab) Injection (Revised January 2008).

Weak cation-exchange chromatography (WCX) analysis of adalimumab hasshown that it has three main basic charge variants (i.e., Lys 0, Lys 1,and Lys 2). These variants, or charged isomers, are the result ofincomplete post-translational cleavage of the C-terminal lysine residueson the heavy chains of the antibody. In addition to the lysine variants,the WCX-10 analysis measures the presence acidic species. These acidicspecies regions (i.e., acidic species), AR1 and AR2, are classified asproduct-related impurities that are relatively acidic when compared tothe lysine variants and elute before the Lys 0 peak in the chromatogram(see, for example, FIG. 1).

As used herein, the term “lysine variant species” refers to an antibody,or antigen-binding portion thereof, comprising heavy chains with eitherzero, one or two C-terminal lysines. For example, the “Lys 0” variantcomprises an antibody, or antigen-binding portion thereof, with heavychains that do not comprise a C-terminal lysine. The “Lys 1” variantcomprises an antibody, or antigen-binding portion thereof, with oneheavy chain that comprises a C-terminal lysine. The “Lys 2” variantcomprises an antibody, or antigen-binding portion thereof, with bothheavy chains comprising a C-terminal lysine. Lysine variants can bedetected by weak cation exchange chromatography, for example, WCX, ofthe expression product of a host cell expressing the antibody, orantigen-binding portion thereof. For example, but not by way oflimitation, FIG. 2 depicts WCX analysis of adalimumab wherein the threelysine variants, as well as two acidic species, are resolved from eachother.

A composition of the invention may comprise more than one lysine variantspecies of an antibody, or antigen-binding portion thereof. For example,in one embodiment, the composition may comprise a Lys 2 variant of anantibody, or antigen-binding portion thereof. The composition maycomprise a Lys 1 variant of an antibody, or antigen-binding portionthereof. The composition may comprise a Lys 0 variant of an antibody, orantigen-binding portion thereof. In another embodiment, the compositionmay comprise both Lys 1 and Lys 2, or Lys 1 and Lys 0, or Lys 2 and Lys0 variants of an antibody, or antigen-binding portion thereof. Inanother embodiment, the composition may comprise all three lysinevariant species, i.e., Lys 0, Lys 1 and Lys 2, of an antibody, orantigen-binding portion thereof.

As used herein, the phrases “antibody resistant to C-terminalprocessing” or “antibody resistant to C-terminal processing by acarboxypeptidase” refer to an antibody, or antigen-binding portionthereof, that is resistant to processing of the C-terminus of its heavychains by a carboxypeptidase enzyme, e.g., carboxypeptidase B orcarboxypeptidase U. An “antibody resistant to C-terminal processing”exhibits decreased removal of a C-terminal lysine of its heavy chains bya carboxypeptidase enzyme, e.g., carboxypeptidase B or carboxypeptidaseU. The antibody, or antigen-binding portion thereof, may be modified asdescribed herein to exhibit decreased removal of a C-terminal lysine ascompared to an antibody, or antigen-binding portion thereof, that hasnot been modified. In one embodiment, the antibody, or antigen-bindingportion thereof, retains both C-terminal lysines (“Lys 2”) and, thus,exhibits no removal (i.e., exhibits no C-terminal processing) of theC-terminal lysines of the heavy chains by a carboxypeptidase. C-terminalprocessing by a carboxypeptidase may be measured using assays that arewell-known in the art including, but not limited to, the peptidaseassays described in the Examples section below.

As used herein, the term “carboxypeptidase” refers to a protease enzymethat hydrolyzes a peptide bond at the carboxy-terminal (“C-terminal”)region of a protein or antibody. Carboxypeptidases are well-known in theart and are involved in post-translational modification of proteins.Specifically, “carboxypeptidase B” (EC 3.4.17.2) refers to acarboxypeptidase that preferentially cleaves positively charged, orbasic, amino acids, such as arginine and lysine from the c-terminus ofproteins and antibodies. “Carboxypeptidase U” or “unstablecarboxypeptidase” (EC 3.4.17.20) refers to a carboxypeptidase that isactivated by thrombin or plasmin during clotting.

The term “modify”, “modifying” or “modified,” as used herein, isintended to refer to changing one or more amino acids in an antibody, orantigen-binding portion thereof. The change can be produced by adding,substituting or deleting an amino acid at one or more positions. Thechange can be produced using standard techniques known in the art anddescribed in more detail herein, such as PCR mutagenesis andsite-directed mutagenesis. In one embodiment, of the invention, theC-terminal three amino acids of the heavy chain sequences of anantibody, or antigen-binding portion thereof, are modified from thenative sequence of proline-glycine-lysine (“PGK”) (SEQ ID NO:10) toinclude a proline between the glycine and lysine, resulting in aC-terminal sequence of proline-glycine-proline-lysine (“PGPK”) (SEQ IDNO:9). For example, the C-terminal three amino acids of the heavy chainsequence of adalimumab (SEQ ID NO:14) can be modified from the nativesequence of proline-glycine-lysine (“PGK”) (see, e.g., SEQ ID NO:14) toinclude a proline between the glycine and lysine, resulting in aC-terminal sequence of proline-glycine-proline-lysine (“PGPK”) (see,e.g., SEQ ID NO:15).

As used herein, the phrase “increased cartilage tissue penetration”refers to the property of an antibody, or antigen-binding portionthereof, of the invention showing increased penetration of cartilagetissue. This property can be measured or determined by, for example,using an in vitro or an in vivo cartilage model. In one embodiment, anantibody, or antigen-binding portion thereof, retains both C-terminallysines (“Lys 2”) and exhibits increased cartilage penetration ascompared to an antibody, or antigen-binding portion thereof, having onlyone C-terminal lysine (“Lys 1”) or no C-terminal lysines (“Lys 0”). Inone embodiment, the antibody, or antigen-binding portion thereof, hasbeen modified to exhibit increased cartilage penetration as compared toan antibody, or antigen-binding portion thereof, that has not beenmodified. Cartilage penetration can be measured using assays that arewell-known in the art including, but not limited to, the assaysdescribed in the Examples section below.

As used herein, the terms “acidic species”, “acidic species regions” and“AR,” refer to the variants of a protein, e.g., an antibody orantigen-binding portion thereof, which are characterized by an acidiccharge. For example, in monoclonal antibody (mAb) preparations, suchacidic species can be detected by various methods, such as, for example,WCX-10 HPLC (a weak cation exchange chromatography), or IEF (isoelectricfocusing).

Acidic species of an antibody include charge variants, structurevariants, and/or fragmentation variants. Exemplary charge variantsinclude, but are not limited to, deamidation variants, afucosylationvariants, methylglyoxal (MGO) variants, glycation variants, and citricacid variants. Exemplary structure variants include, but are not limitedto, glycosylation variants and acetonation variants. Exemplaryfragmentation variants include any truncated protein species from thetarget molecule due to dissociation of peptide chain, enzymatic and/orchemical modifications, including, but not limited to, Fc and Fabfragments, fragments missing a Fab, fragments missing a heavy chainvariable domain, C-terminal truncation variants, variants with excisionof N-terminal Asp in the light chain, and variants having N-terminaltruncation of the light chain. Other acidic species variants includevariants containing unpaired disulfides, host cell proteins, and hostnucleic acids, chromatographic materials, and media components.

In certain embodiments, a protein composition can comprise more than onetype of acidic species variant. For example, but not by way oflimitation, the total acidic species can be divided based onchromatographic residence time. For example, as disclosed in FIG. 1, thetotal acidic species associated with the expression of adalimumab may bedivided into a first acidic species region (AR1) and a second acidicspecies region (AR2). AR1 may comprise, for example, charge variantssuch as deamidation variants, MGO modified species, glycation variants,and citric acid variants, structural variants such as glycosylationvariants and acetonation variants, and/or fragmentation variants. Otheracidic variants such as host cells and unknown species may also bepresent. AR2 may comprise, for example, charge variants such asglycation variants and deamidation variants.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., TNFα) and still contain at least one heavy chain. It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Such antibody embodiments mayalso be bispecific, dual specific, or multi-specific formats;specifically binding to two or more different antigens. Examples ofbinding fragments encompassed within the term “antigen-binding portion”of an antibody include a Fv fragment consisting of the VL and VH domainsof a single arm of an antibody or a halfbody (as described in, forexample, PCT Publication No. WO12/088,302, the entire contents of whichare incorporated herein by reference). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger, P., et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al.(1994) Structure 2:1121-1123). Such antibody binding portions are knownin the art (Kontermann and Dubel eds., Antibody Engineering (2001)Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

The term “antibody construct” as used herein refers to a polypeptidecomprising one or more of the antigen binding portions of the inventionlinked to a linker polypeptide or an immunoglobulin constant domain.Linker polypeptides comprise two or more amino acid residues joined bypeptide bonds and are used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see e.g., Holliger,P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constantdomain refers to a heavy or light chain constant domain. Human IgG heavychain and light chain constant domain amino acid sequences are known inthe art.

An antibody or antigen-binding portion thereof may be part of a largerimmunoadhesion molecule, formed by covalent or noncovalent associationof the antibody or antibody portion with one or more other proteins orpeptides. Examples of such immunoadhesion molecules include use of thestreptavidin core region to make a tetrameric scFv molecule (Kipriyanov,S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and useof a cysteine residue, a marker peptide and a C-terminal polyhistidinetag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M.,et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such asFab and F(ab′)₂ fragments, can be prepared from whole antibodies usingconventional techniques, such as papain or pepsin digestion,respectively, of whole antibodies. Moreover, antibodies, antibodyportions and immunoadhesion molecules can be obtained using standardrecombinant DNA techniques, as described herein.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds human TNFα). An isolated antibody that specifically binds TNFαmay, however, have cross-reactivity to other antigens, such as the TNFαmolecules from other species. Alternatively, an isolated antibody, orantigen-binding portion thereof, may not cross-react with the TNFαmolecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell. In another embodiment, the humanmonoclonal antibodies are produced by phage display technologies asdescribed, for example, in the Examples section below.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library(Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and HighsmithW. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J.W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000)Immunology Today 21:371-378), antibodies isolated from an animal (e.g.,a mouse) that is transgenic for human immunoglobulin genes (see e.g.,U.S. Pat. No. 6,713,610; Taylor, L. D., et al. (1992) Nucl. Acids Res.20:6287-6295; Kellermann S-A., and Green L. L. (2002) Current Opinion inBiotechnology 13:593-597; Little M. et al (2000) Immunology Today21:364-370) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which human CDR sequences are introduced intonon-human VH and VL sequences to replace the corresponding nonhuman CDRsequences. Such antibodies were generated by obtaining murine anti-TNFαmonoclonal antibodies using traditional hybridoma technology followed byhumanization using in vitro genetic engineering.

The term “antibody mimetic” or “antibody mimic” is intended to refer tomolecules capable of mimicking an antibody's ability to bind an antigen,but which are not limited to native antibody structures. Examples ofsuch antibody mimetics include, but are not limited to, Adnectins (i.e.,fibronectin based binding molecules), Affibodies, DARPins, Anticalins,Avimers, and Versabodies all of which employ binding structures that,while they mimic traditional antibody binding, are generated from andfunction via distinct mechanisms. The embodiments of the instantinvention, as they are directed to antibodies, or antigen bindingportions thereof, also apply to the antibody mimetics described above.

As used herein, “isotype” refers to an antibody class (e.g., IgM orIgG₁) that is encoded by the heavy chain constant region genes.

The terms “Kabat numbering”, “Kabat definitions and “Kabat labeling” areused interchangeably herein. These terms, which are recognized in theart, refer to a system of numbering amino acid residues which are morevariable (i.e., hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody or nucleic acid sequence providing or encoding at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% of the amino acid sequences ofone or more of the framework regions. In some embodiments, the term“acceptor” refers to the antibody amino acid or nucleic acid sequenceproviding or encoding the constant region(s). In yet another embodiment,the term “acceptor” refers to the antibody amino acid or nucleic acidsequence providing or encoding one or more of the framework regions andthe constant region(s). In a specific embodiment, the term “acceptor”refers to a human antibody amino acid or nucleic acid sequence thatprovides or encodes at least 80%, or, at least 85%, at least 90%, atleast 95%, at least 98%, or 100% of the amino acid sequences of one ormore of the framework regions. In accordance with this embodiment, anacceptor may contain at least 1, at least 2, at least 3, least 4, atleast 5, or at least 10 amino acid residues that does (do) not occur atone or more specific positions of a human antibody. An acceptorframework region and/or acceptor constant region(s) may be, e.g.,derived or obtained from a germline antibody gene, a mature antibodygene, a functional antibody (e.g., antibodies well-known in the art,antibodies in development, or antibodies commercially available).

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems, although preferred embodiments useKabat or Chothia defined CDRs.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia etal., J. Mol. Biol. 227:799 (1992), both are incorporated herein byreference). According to Chothia et al., critical portions of the CDRsof many antibodies have nearly identical peptide backbone confirmationsdespite great diversity at the level of amino acid sequence. Eachcanonical structure specifies primarily a set of peptide backbonetorsion angles for a contiguous segment of amino acid residues forming aloop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In one embodiment, the donorantibody is an antibody from a species different from the antibody fromwhich the framework regions are obtained or derived. In the context of ahumanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, CDR-L2,and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain)also divide the framework regions on the light chain and the heavy chaininto four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in whichCDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, andCDR3 between FR3 and FR4. Without specifying the particular sub-regionsas FR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain acceptor sequences are known in theart.

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med. Biol. 484:13-30(2001)). One of the advantages of germline antibody genes stems from therecognition that germline antibody genes are more likely than matureantibody genes to conserve essential amino acid sequence structurescharacteristic of individuals in the species, hence less likely to berecognized as from a foreign source when used therapeutically in thatspecies.

As used herein, the term “key” residues refer to certain residues withinthe variable region that have more impact on the binding specificityand/or affinity of an antibody, in particular a humanized antibody. Akey residue includes, but is not limited to, one or more of thefollowing: a residue that is adjacent to a CDR, a potentialglycosylation site (can be either N- or O-glycosylation site), a rareresidue, a residue capable of interacting with the antigen, a residuecapable of interacting with a CDR, a canonical residue, a contactresidue between heavy chain variable region and light chain variableregion, a residue within the Vernier zone, and a residue in the regionthat overlaps between the Chothia definition of a variable heavy chainCDR1 and the Kabat definition of the first heavy chain framework.

As used herein, the term “humanized antibody” is an antibody or avariant, derivative, analog or fragment thereof which binds to anantigen of interest and which comprises a framework (FR) region havingsubstantially the amino acid sequence of a human antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% identical to theamino acid sequence of a non-human antibody CDR. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains (Fab, Fab′, F(ab′)₂, FabC, Fv) in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin(i.e., donor antibody) and all or substantially all of the frameworkregions are those of a human immunoglobulin consensus sequence. Ahumanized antibody may also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG 1, IgG2, IgG3 and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. Such mutations, however, willnot be extensive. Usually, at least 80%, at least 85%, at least 90%, andat least 95% of the humanized antibody residues will correspond to thoseof the parental FR and CDR sequences. As used herein, the term“consensus framework” refers to the framework region in the consensusimmunoglobulin sequence. As used herein, the term “consensusimmunoglobulin sequence” refers to the sequence formed from the mostfrequently occurring amino acids (or nucleotides) in a family of relatedimmunoglobulin sequences (See e.g., Winnaker, From Genes to Clones(Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.If two amino acids occur equally frequently, either can be included inthe consensus sequence.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

The term “multivalent binding protein” is used in this specification todenote a binding protein comprising two or more antigen binding sites.The multivalent binding protein is engineered to have the three or moreantigen binding sites, and is generally not a naturally occurringantibody. The term “multispecific binding protein” refers to a bindingprotein capable of binding two or more related or unrelated targets.Dual variable domain (DVD) binding proteins as used herein, are bindingproteins that comprise two or more antigen binding sites and aretetravalent or multivalent binding proteins. Such DVDs may bemonospecific, i.e. capable of binding one antigen or multispecific, i.e.capable of binding two or more antigens. DVD binding proteins comprisingtwo heavy chain DVD polypeptides and two light chain DVD polypeptidesare referred to a DVD Ig. Each half of a DVD Ig comprises a heavy chainDVD polypeptide, and a light chain DVD polypeptide, and two antigenbinding sites. Each binding site comprises a heavy chain variable domainand a light chain variable domain with a total of 6 CDRs involved inantigen binding per antigen binding site.

As used herein, the term “neutralizing” refers to neutralization ofbiological activity of TNFα. A neutralizing binding protein is aneutralizing antibody whose binding to TNFα and/or a mutant TNFα proteinresults in inhibition of a biological activity of TNFα and/or the mutantTNFα. The neutralizing binding protein binds TNFα and/or a mutant TNFαprotein and reduces a biologically activity of TNFα and/or a mutant TNFαprotein by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,95% or more Inhibition of a biological activity of TNFα and/or a mutantTNFα protein by a neutralizing binding protein can be assessed bymeasuring one or more indicators of TNFα and/or mutant TNFα biologicalactivity well known in the art.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, for example, ananti-TNFα antibody that binds to a TNFα antigen and/or the neutralizingpotency of an antibody, for example, an anti-TNFα antibody whose bindingto TNFα inhibits the biological activity of TNFα.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. In certain embodiments, an antibody is said to specificallybind an antigen when it preferentially recognizes its target antigen ina complex mixture of proteins and/or macromolecules.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin.51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody with another moiety,e.g., TNFα, mean an interaction that is dependent upon the presence of aparticular structure (e.g., an antigenic determinant or epitope) on themoiety, e.g., TNFα. For example, an antibody recognizes and binds to aspecific protein structure rather than to proteins, generally. If anantibody is specific for epitope “A”, the presence of a moleculecontaining epitope A (or free, unlabeled A), in a reaction containinglabeled “A” and the antibody, will reduce the amount of labeled A boundto the antibody.

As used herein, an antibody that “binds” or “specifically binds” to anantigen, e.g., TNFα, is intended to refer to an antibody, orantigen-binding portion thereof, that specifically binds to the antigen.The term “K_(on)” (also “Kon”, “kon”), as used herein, is intended torefer to the on rate constant for association of a binding protein ofthe invention (e.g., an antibody of the invention) to an antigen to forman association complex, e.g., antibody/antigen complex, as is known inthe art. The “K_(on)” also is known by the terms “association rateconstant”, or “ka”, as used interchangeably herein. This value indicatesthe binding rate of an antibody to its target antigen or the rate ofcomplex formation between an antibody and antigen as is shown by theequation below:Antibody (“Ab”)+Antigen (“Ag”)→Ab-Ag.

The term “K_(off)” (also “Koff”, “koff”), as used herein, is intended torefer to the off rate constant for dissociation, or “dissociation rateconstant”, of a binding protein of the invention (e.g., an antibody ofthe invention) from an association complex (e.g., an antibody/antigencomplex) as is known in the art. This value indicates the dissociationrate of an antibody from its target antigen or separation of Ab-Agcomplex over time into free antibody and antigen as shown by theequation below:Ab+Ag←Ab-Ag.

The term “K_(D)” (also “K_(d)”), as used herein, is intended to refer tothe “equilibrium dissociation constant”, and refers to the valueobtained in a titration measurement at equilibrium, or by dividing thedissociation rate constant (Koff) by the association rate constant(Kon). The association rate constant (Kon), the dissociation rateconstant (Koff), and the equilibrium dissociation constant (K are usedto represent the binding affinity of an antibody to an antigen. Methodsfor determining association and dissociation rate constants are wellknown in the art. Using fluorescence-based techniques offers highsensitivity and the ability to examine samples in physiological buffersat equilibrium. Other experimental approaches and instruments such as aBIAcore® (biomolecular interaction analysis) assay can be used (e.g.,instrument available from BIAcore International AB, a GE Healthcarecompany, Uppsala, Sweden). Additionally, a KinExA® (Kinetic ExclusionAssay) assay, available from Sapidyne Instruments (Boise, Id.) can alsobe used.

The term “labeled binding protein” as used herein, refers to a proteinwith a label incorporated that provides for the identification of thebinding protein. In one aspect, the label is a detectable marker, e.g.,incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods).Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, luciferase, alkaline phosphatase);chemiluminescent markers; biotinyl groups; predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags); and magnetic agents, such as gadoliniumchelates. Representative examples of labels commonly employed forimmunoassays include moieties that produce light, e.g., acridiniumcompounds, and moieties that produce fluorescence, e.g., fluorescein.Other labels are described herein. In this regard, the moiety itself maynot be detectably labeled but may become detectable upon reaction withyet another moiety. Use of the term “detectably labeled” is intended toencompass the latter type of detectable labeling.

The term “antibody conjugate” refers to a binding protein, such as anantibody, linked, e.g., chemically linked, to a second chemical moiety,such as a therapeutic or cytotoxic agent. The term “agent” is usedherein to denote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materials.In one aspect, the therapeutic or cytotoxic agents include, but are notlimited to, pertussis toxin, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof.

The term “polynucleotide” as referred to herein, means a polymeric formof two or more nucleotides, either ribonucleotides or deoxynucleotidesor a modified form of either type of nucleotide. The term includessingle and double stranded forms of DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide (e.g., of genomic, cDNA, or synthetic origin, or somecombination thereof) that, by virtue of its origin, is not associatedwith all or a portion of a polynucleotide with which the “isolatedpolynucleotide” is found in nature; is operably linked to apolynucleotide to which it is not linked in nature; or does not occur innature as part of a larger sequence.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences which are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Protein constructs of the presentinvention may be expressed, and purified using expression vectors andhost cells known in the art, including expression cassettes, vectors,recombinant host cells and methods for the recombinant expression andproteolytic processing of recombinant polyproteins and pre-proteins froma single open reading frame (e.g., WO 2007/014162, the entire contentsof which are incorporated herein by reference).

“Transformation”, as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which exogenous DNA has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell, but, to the progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein. Hostcells may include prokaryotic and eukaryotic cells selected from any ofthe Kingdoms of life. In one aspect, eukaryotic cells include protist,fungal, plant and animal cells. In a particular aspect, host cellsinclude but are not limited to the prokaryotic cell line E. Coli;mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; andthe fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

The terms “regulate” and “modulate” as used interchangeably, and referto a change or an alteration in the activity of a molecule of interest(e.g., the biological activity of TNFα). Modulation may be an increaseor a decrease in the magnitude of a certain activity or function of themolecule of interest. Exemplary activities and functions of a moleculeinclude, but are not limited to, binding characteristics, enzymaticactivity, cell receptor activation, and signal transduction.

As used herein, the term “effective amount” refers to the amount of atherapy which is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof, prevent theadvancement of a disorder, cause regression of a disorder, prevent therecurrence, development, onset or progression of one or more symptomsassociated with a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent). In one embodiment, an “effectiveamount” refers to the amount of an antibody, or antigen-binding portionthereof, of the invention, e.g., an anti-TNFα antibody, orantigen-binding portion thereof, that is sufficient to treat a disorderin which TNFα activity is detrimental.

As used herein, the term “a disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of TNFα in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which TNFαactivity is detrimental is a disorder in which inhibition of TNFαactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of TNFα in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofTNFα in serum, plasma, synovial fluid, etc. of the subject), which canbe detected, for example, using an anti-TNFα antibody. There arenumerous examples of disorders in which TNFα activity is detrimentaldescribed in more detail below. In one embodiment, the disorder in whichTNFα activity is detrimental is selected from the group consisting of anautoimmune disorder, an intestinal disorder, and a skin disease. In oneembodiment, the autoimmune disorder is selected from the groupconsisting of rheumatoid arthritis, rheumatoid spondylitis,osteoarthritis, gouty arthritis, an allergy, multiple sclerosis,psoriatic arthritis, autoimmune diabetes, autoimmune uveitis, nephroticsyndrome and juvenile rheumatoid arthritis. In one embodiment, theintestinal disorder is Crohn's disease. In one embodiment, the skindisease is psoriasis. The use of TNFα antibodies and antibody portionsobtained using methods of the invention for the treatment of specificdisorders is discussed in detail further below.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Antibodies, and Antigen-Binding Portions Thereof

The present invention provides antibodies, and antigen-binding portionsthereof, having mutations to the C-terminal residues of their heavychains that result in a decrease in the C-terminal processing of theheavy chains, thereby leading to more efficacious and stable antibodiesand antibody compositions. The present invention also providescompositions comprising antibodies, and antigen-binding portionsthereof, which exhibit decreased levels of C-terminal processing oflysines on the heavy chains. In one embodiment, the antibody, orantigen-binding portion thereof, is an anti-TNFα antibody, orantigen-binding portion thereof, such as adalimumab, modified accordingto the methods of the invention.

Exemplary antibodies are provided herein. The features of such exemplaryantibodies are set forth in the herein and in the Sequence Listing,Figures, Tables and Examples.

1. C-Terminal Lysines

The present invention is based, at least in part, on the discovery thatthe C-terminal lysines of antibodies in pharmaceutical compositions canbe lost during both the purification process and storage of the finalcomposition as well as in vivo, resulting in compositions comprisingindividual antibody species that can vary at their C-terminus as towhether a lysine residue is present. This heterogeneity may lead todecreased efficacy and stability of the drug product. The inventors ofthe instant application have surprisingly discovered that modifying anantibody, or antigen-binding portion thereof, comprising a C-terminalheavy chain sequence of proline-glycine-lysine (“PGK”) by inserting aproline between the glycine and lysine (proline-glycine-proline-lysine,or “PGPK”) (SEQ ID NO:9) prevents the C-terminal processing of theantibody heavy chain and leads to more efficacious and stable antibodycompositions. Without intending to be limited by theory, it is believedthat these modifications make the antibody resistant to processing by aC-terminal carboxypeptidase (e.g., carboxypeptidase B orcarboxypeptidase U).

As used herein, the term “modify”, “modifying” or “modified,” isintended to refer to changing one or more amino acids in an antibody, orantigen-binding portion thereof. The change can be produced by adding,substituting or deleting an amino acid at one or more positions. Thechange can be produced using standard techniques known in the art anddescribed in more detail herein, such as PCR mutagenesis andsite-directed mutagenesis. In one embodiment, of the invention, theC-terminal three amino acids of the heavy chain sequences of anantibody, or antigen-binding portion thereof, are modified from thenative sequence of proline-glycine-lysine (“PGK”) (SEQ ID NO:10) toinclude a proline between the glycine and lysine, resulting in aC-terminal sequence of proline-glycine-proline-lysine (“PGPK”) (SEQ IDNO:9). In another embodiment, the C-terminal three amino acids of theheavy chain sequence of adalimumab are modified from the native sequenceof proline-glycine-lysine (“PGK”) (see, e.g., SEQ ID NO:14) to include aproline between the glycine and lysine, resulting in a C-terminalsequence of proline-glycine-proline-lysine (“PGPK”) (see, e.g., SEQ IDNO:15).

As used herein, the term “lysine variant species” refers to an antibody,or antigen-binding portion thereof, comprising heavy chains with eitherzero, one or two C-terminal lysines. For example, the “Lys 0” variantcomprises an antibody, or antigen-binding portion thereof, with heavychains that do not comprise a C-terminal lysine. The “Lys 1” variantcomprises an antibody, or antigen-binding portion thereof, with oneheavy chain that comprises a C-terminal lysine. The “Lys 2” variantcomprises an antibody with both heavy chains comprising a C-terminallysine. Lysine variants can be detected, for example, by weak cationexchange chromatography (WCX) of the expression product of a host cellexpressing the antibody, or antigen-binding portion thereof. Forexample, but not by way of limitation, FIG. 2 depicts WCX analysis ofadalimumab wherein the three lysine variants, as well as two acidicspecies, are resolved from each other.

A composition of the invention may comprise more than one lysine variantspecies of an antibody, or antigen-binding portion thereof. For example,in one embodiment, the composition may comprise a Lys 2 variant of anantibody, or antigen-binding portion thereof. The composition maycomprise a Lys 1 variant of an antibody, or antigen-binding portionthereof. The composition may comprise a Lys 0 variant of an antibody, orantigen-binding portion thereof. In another embodiment, the compositionmay comprise both Lys 1 and Lys 2 variants, or Lys 0 and Lys 2 variants,or Lys 0 and Lys 1 variants of an antibody, or antigen-binding portionthereof. In another embodiment, the composition may comprise all threelysine variant species, i.e., Lys 0, Lys 1 and Lys 2, of an antibody, orantigen-binding portion thereof.

In one embodiment, the invention comprises a composition comprising anantibody, or antigen-binding portion thereof, wherein the compositioncomprises less than about 50% lysine variant species that lack aC-terminal lysine (Lys 0). In another embodiment, the compositioncomprises less than about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%,40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%,26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% lysine variantspecies that lack a C-terminal lysine (“Lys 0”). In another embodiment,the composition comprises about 50% to about 0%, about 40% to about 10%,about 30% to about 20%, about 40% to about 20%, or about 30% to about15% lysine variant species that lack a C-terminal lysine (Lys 0). In oneembodiment, the composition comprises 0% lysine variant species thatlack a C-terminal lysine (Lys 0). As used herein, the percent lysinevariant species in the composition refers to the weight of the specificlysine variant species in a sample in relation to the weight of thetotal lysine variant species sum (i.e., the sum of Lys 0, Lys 1 and Lys2) contained in the sample or composition. For example, the percentlysine variant species can be calculated using weak cation exchangechromatography such as WCX-10, as described herein.

In another embodiment, the composition comprises less than about 25%lysine variant species that have one C-terminal lysine (Lys 1). Inanother embodiment, the composition comprises less than about 24%, 23%,22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2% or 1% lysine variant species that have oneC-terminal lysine (Lys 1). In another embodiment, the compositioncomprises about 25% to about 0%, about 20% to about 5%, about 15% toabout 10%, about 20% to about 10%, about 15% to about 5%, or about 25%to about 5% lysine variant species that have one C-terminal lysine (Lys1). In one embodiment, the composition comprises 0% lysine variantspecies that have one C-terminal Lysine (Lys 1).

In another embodiment, the composition comprises at least about 70%lysine variant species that have two C-terminal lysines (Lys 2). Inanother embodiment, the composition comprises at least about 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%lysine variant species that have two C-terminal lysines (Lys 2). In oneembodiment, the composition comprises about 70% to about 100%, about 70%to about 90%, about 70% to about 80%, about 80% to about 100%, about 85%to about 100%, about 90% to about 100%, about 95% to about 100%, about80% to about 90%, about 85% to about 95%, about 75% to about 85%, orabout 97% to about 100% lysine variant species that have two C-terminallysines (Lys 2). In one embodiment, the composition comprises 100%lysine variant species that have two C-terminal lysines (Lys 2).

In one embodiment, the composition may comprise less than about 10%acidic species, wherein the acidic species comprise a first acidicspecies region (AR1) and a second acidic species region (AR2). Inanother embodiment, the composition may comprise less than about 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% acidic species. In another embodiment,the composition may comprise about 3% acidic species. In anotherembodiment, the composition may comprise less than about 1% AR1. Inanother embodiment, the composition may comprise about 0% AR1. Inanother embodiment, the composition may comprise less than about 5%, 4%,3%, 2%, 1% or 0% AR2. In another embodiment, the composition maycomprise about 0% AR1 and about 3% AR2. In yet another embodiment, thecomposition may comprise about 1% AR1 and about 4% AR2. As used herein,the percent AR in the composition refers to the weight of the acidicspecies in a sample in relation to the weight of the total antibodiescontained in the sample. For example, the percent AR can be calculatedusing weak cation exchange chromatography such as WCX-10, as describedherein.

In one embodiment of the invention, the antibody, or antigen-bindingportion thereof, containing a PGPK modification is an anti-TNFαantibody, or antigen-binding portion thereof. For example, the inventioncomprises an anti-TNFα antibody, or antigen-binding portion thereof,comprising the full-length heavy and light chain sequences ofadalimumab, except that the C-terminal three amino acids of the heavychain sequences are modified from the native IgG₁ sequence of PGK toinclude a proline between the glycine and lysine to result in aC-terminal sequence of PGPK. In certain embodiments, the instantinvention is directed to an anti-TNFα antibody, or antigen-bindingportion thereof, comprising the full-length heavy and light chainsequences of adalimumab, but which comprise a PGPK C-terminal sequenceand at least one additional sequence modification to the heavy or lightchain sequences. In certain embodiments, the additional sequencemodification, or modifications, can include conservative ornon-conservative substitutions, insertions, and/or deletions.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody can be tested for retainedfunction using the functional assays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package, usingeither a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

In other embodiments, the present invention is also directed to nucleicacid sequences, such as DNA sequences and/or RNA sequences, encoding theantibodies, and antigen-binding portions thereof, of the presentinvention. For example, but not by way of limitation, the nucleic acidsequences of the present invention would include nucleic acid sequencesencoding the full length heavy or light chain amino acid sequences ofadalimumab, except that the C-terminal three amino acids of the heavychain sequences is modified from the native antibody sequence of PGK toinclude a proline between the glycine and lysine to result in aC-terminal sequence of PGPK. Thus, in certain embodiments the nucleicacid sequences of the present invention include the addition of a CCC,CCU/T, CCA, or CCG codon (where U is employed in the codon if thenucleic acid is RNA and T is employed if the codon is DNA) between thesequences coding for the C-terminal lysine and the glycine immediatelypreceding it. In certain embodiments, the nucleic acids of the instantinvention will comprise a sequence encoding a heavy chain C-terminalsequence of PGPK and at least one additional sequence modification tothe heavy or light chain sequences. In certain embodiments, theadditional sequence modification, or modifications, can includeconservative or non-conservative substitutions, insertions, and/ordeletions.

2. Variable Regions

The antigen-binding portion of an antibody comprises one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., human TNFα, or a portion thereof). It has been shownthat the antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, the light chain variable domain (VL) and the heavy chainvariable domain (VH), are encoded by separate genes, they can be joined,using recombinant methods, by a synthetic linker that enables them to bemade as a single protein chain in which the VL and VH regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for function in the same manner as are intactantibodies. Antigen-binding portions can be produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intactimmunoglobulins.

The antibodies of the present invention comprise at least one antigenbinding domain. In one embodiment, the antibodies of the invention areanti-TNFα antibodies, or antigen-binding portions thereof.

In certain embodiments, the antibodies are human or chimeric antibodiesor antibody fragments. In certain embodiments, the antibodies are humanantibodies or antibody fragments. In other embodiments, the antibodiesare human or chimeric antibodies or antibody fragments that bind tohuman TNFα and inhibit TNFα activity.

In certain embodiments, the antibodies comprise a VH and/or VL domainthat has a given percent identify to at least one of the VH and/or VLsequences disclosed herein. As used herein, the term “percent (%)sequence identity”, also including “homology” is defined as thepercentage of amino acid residues or nucleotides in a candidate sequencethat are identical with the amino acid residues or nucleotides in thereference sequences after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Optimal alignment of the sequences for comparison may beproduced, besides manually, by means of the local homology algorithm ofSmith and Waterman, 1981, Ads App. Math. 2, 482, by means of the localhomology algorithm of Neddleman and Wunsch, 1970, J. MoI. Biol. 48, 443,by means of the similarity search method of Pearson and Lipman, 1988,Proc. Natl Acad. Sci. USA 85, 2444, or by means of computer programswhich use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N andTFASTA in Wisconsin Genetics Software Package, Genetics Computer Group,575 Science Drive, Madison, Wis.).

The antibodies of the invention containing a PGPK modification maycomprise, or have, a heavy chain variable region (HCVR) amino acidsequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more, identity to the amino acid sequence of SEQ ID NO:2.

The antibodies of the invention containing a PGPK modification maycomprise a HCVR amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 amino acid residue substitutions have been made relative to SEQ IDNO:2. The substitutions may be conservative amino acid substitutions.These antibodies may have at least two or more (e.g., at least 3, 4, 5,6, 7, 8, 9, 10 or more) of the biological characteristics describedherein.

The antibodies of the invention containing a PGPK modification maycomprise, or have, a light chain variable region (LCVR) domain aminoacid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or more identity to SEQ ID NO:1.

The antibodies of the invention containing a PGPK modification maycomprise, or have, a LCVR amino acid sequence in which 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acid residue substitutions have been made relativeto SEQ ID NO:1. In certain embodiments, the substitutions areconservative amino acid substitutions. These antibodies have at leasttwo or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of thebiological characteristics described herein.

In a specific embodiment, the antibodies, or antigen-binding portionsthereof, of the invention containing a PGPK modification may comprise,or have, a HCVR amino acid sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2, and a LCVRamino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identity to SEQ ID NO:1. These antibodies may haveat least two more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) ofthe TNFα biological characteristics described herein.

In certain embodiments, the antibodies containing a PGPK modificationmay comprise, or have, a HCVR amino acid sequence in which 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been maderelative to SEQ ID NO:2, and a LCVR amino acid sequence in which 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have beenmade relative to SEQ ID NO:1. These antibodies may have at least twomore (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the TNFαbiological characteristics described herein.

3. Complementarity Determining Regions (CDRs)

Although the variable domain (VH and VL) comprises the antigen-bindingregion, the variability is not evenly distributed through the variabledomains of antibodies. It is concentrated in segments calledComplementarity Determining Regions (CDRs), both in the light chain (VLor VK) and the heavy chain (VH) variable domains. The more highlyconserved portions of the variable domains are called the frameworkregions (FR). The variable domains of native heavy and light chains eachcomprise four FR, largely adopting a β-sheet configuration, connected bythree CDRs, which form loops connecting, and in some cases forming partof, the β-sheet structure. The CDRs in each chain are held together inclose proximity by the FR and, with the CDRs from the other chain,contribute to the formation of the antigen-binding site of antibodies(see, Kabat et al., Supra). The three CDRs of the heavy chain aredesignated CDR-H1, CDR-H2, and CDR-H3, and the three CDRs of the lightchain are designated CDR-L1, CDR-L2, and CDR-L3. The Kabat numberingsystem is used herein. As such, CDR-H1 begins at approximately aminoacid 31 (i.e., approximately 9 residues after the first cysteineresidue), includes approximately 5-7 amino acids, and ends at the nexttyrosine residue. CDR-H2 begins at the fifteenth residue after the endof CDR-H1, includes approximately 16-19 amino acids, and ends at thenext arginine or lysine residue. CDR-H3 begins at approximately thethirty third amino acid residue after the end of CDR-H2; includes 3-25amino acids; and ends at the sequence W-G-X-G, where X is any aminoacid. CDR-L1 begins at approximately residue 24 (i.e., following acysteine residue); includes approximately 10-17 residues; and ends atthe next tyrosine residue. CDR-L2 begins at approximately the sixteenthresidue after the end of CDR-L1 and includes approximately 7 residues.CDR-L3 begins at approximately the thirty third residue after the end ofCDR-L2; includes approximately 7-11 residues and ends at the sequenceF-G-X-G, where X is any amino acid. Note that CDRs vary considerablyfrom antibody to antibody (and by definition will not exhibit homologywith the Kabat consensus sequences).

The antibodies of the invention comprise at least one antigen bindingdomain that comprises at least one complementarity determining region(CDR1, CDR2 and CDR3). In one embodiment, the antibodies comprise a VHthat comprises at least one VH CDR (e.g., CDR-H1, CDR-H2 or CDR-H3). Inanother embodiment, the antibodies comprise a VL that comprises at leastone VL CDR (e.g., CDR-L1, CDR-L2 or CDR-L3).

In certain embodiments, the antibodies of the invention containing aPGPK modification may comprise a combination of any CDR-H1 sequencedisclosed herein; any CDR-H2 sequence disclosed herein; any CDR-H3sequence disclosed herein; any CDR-L1 sequence disclosed herein; anyCDR-L2 sequence disclosed herein; and any CDR-L3 sequence disclosedherein. In one embodiment, the antibody, or antigen-binding portionthereof, is an anti-TNFα antibody, or antigen-binding portion thereof.In certain embodiments, the antibody is an antibody fragment. In certainembodiments, the antibody is a human, humanized or chimeric antibody.

In certain embodiments, the anti-TNFα antibodies, or antigen-bindingportions thereof, of the invention containing a PGPK modification maycomprise, or have,

(a) a VH CDR1 having an amino acid sequence comprising SEQ ID NO:8,

(b) a VH CDR2 having an amino acid sequence comprising SEQ ID NO:6and/or

(c) a VH CDR3 having an amino acid sequence comprising SEQ ID NO:4.

The anti-TNFα antibodies, or antigen-binding portions thereof, of theinvention containing a PGPK modification may comprise, or have,

(a) a VH CDR1 having an amino acid sequence comprising SEQ ID NO:7,

(b) a VH CDR2 having an amino acid sequence comprising SEQ ID NO:5and/or

(c) a VH CDR3 having an amino acid sequence comprising SEQ ID NO:3.

a. CDR3s

It is well known in the art that VH CDR3 and VL CDR3 domains play animportant role in the binding specificity/affinity of an antibody for anantigen (Xu and Davis, Immunity, 13: 37-45, 2000).

Accordingly, in one embodiment, the antibodies, or antigen-bindingportions thereof, containing a PGPK modification may comprise or have aVH CDR3 having an amino acid sequence identical to or comprising 1, 2,or 3 amino acid residue substitutions relative to SEQ ID NO:4. In oneembodiment, the antibody may be an anti-TNFα antibody, orantigen-binding portion thereof. These anti-TNFα antibodies, orantigen-binding portions thereof, may have at least two or more (e.g.,3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristicsdescribed herein.

In another embodiment, the antibodies, or antigen-binding portionsthereof, containing a PGPK modification may comprise or have a VL CDR3having an amino acid sequence identical to or comprising 1, 2, or 3amino acid residue substitutions relative to SEQ ID NO:3. In oneembodiment, the antibody may be an anti-TNFα antibody, orantigen-binding portion thereof. These anti-TNFα antibodies, orantigen-binding portions thereof, may have at least two or more (e.g.,3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristicsdescribed herein.

In another embodiment, the anti-TNFα antibody, or antigen-bindingportion thereof, containing a PGPK modification may comprise or have:

(a) a VH CDR3 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:4, and

(b) a VL CDR3 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:3.

The invention contemplates antibodies, and antigen-binding antibodyfragments thereof, having any combination of the foregoing VH and VLCDR1s. For example, antibodies containing a PGPK modification maycomprise:

(a) a VH CDR3 having a sequence identical to SEQ ID NO:4,

(b) a VL CDR3 having an amino acid sequence comprising one amino acidsubstitution relative to SEQ ID NO:3. All other combinations aresimilarly contemplated.

The present invention encompasses antibodies comprising amino acids in asequence that is substantially the same as an amino acid sequencedescribed herein. Amino acid sequences that are substantially the sameas the sequences described herein include sequences comprisingconservative amino acid substitutions, as well as amino acid deletionsand/or insertions. A conservative amino acid substitution refers to thereplacement of a first amino acid by a second amino acid that haschemical and/or physical properties (e.g., charge, structure, polarity,hydrophobicity/hydrophilicity) that are similar to those of the firstamino acid. Conservative substitutions include replacement of one aminoacid by another within the following groups: lysine (K), arginine (R)and histidine (H); aspartate (D) and glutamate (E); asparagine (N),glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D andE; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) andglycine (G); F, W and Y; C, S and T. Similarly contemplated is replacinga basic amino acid with another basic amino acid (e.g., replacementamong Lys, Arg, His), replacing an acidic amino acid with another acidicamino acid (e.g., replacement among Asp and Glu), replacing a neutralamino acid with another neutral amino acid (e.g., replacement among Ala,Gly, Ser, Met, Thr, Leu, Ile, Asn, Gln, Phe, Cys, Pro, Trp, Tyr, Val).

The foregoing applies equally to anti-TNFα antibodies and antibodyfragments of the invention. Antibodies and antibody fragments having anyone or more of the foregoing functional and structural characteristicsare contemplated.

4. Framework Regions

The variable domains of the heavy and light chains each comprise fourframework regions (FR1, FR2, FR3, FR4), which are the more highlyconserved portions of the variable domains. The four FRs of the heavychain are designated FR-H1, FR-H2, FR-H3 and FR-H4, and the four FRs ofthe light chain are designated FR-L1, FR-L2, FR-L3 and FR-L4. The Kabatnumbering system is used herein, See Table 1, Kabat et al., Supra. Assuch, FR-H1 begins at position 1 and ends at approximately amino acid30, FR-H2 is approximately from amino acid 36 to 49, FR-H3 isapproximately from amino acid 66 to 94 and FR-H4 is approximately aminoacid 103 to 113. FR-L1 begins at amino acid 1 and ends at approximatelyamino acid 23, FR-L2 is approximately from amino acid 35 to 49, FR-L3 isapproximately from amino acid 57 to 88 and FR-L4 is approximately fromamino acid 98 to 107. In certain embodiments the framework regions maycontain substitutions according to the Kabat numbering system, e.g.,insertion at 106A in FR-L1. In addition to naturally occurringsubstitutions, one or more alterations (e.g., substitutions) of FRresidues may also be introduced in an anti-TNFα antibody. In certainembodiments, these result in an improvement or optimization in thebinding affinity of the antibody for TNFα, for example one or more ofhuman, mouse, or cynomolgous TNFα. Examples of framework region residuesto modify include those which non-covalently bind antigen directly (Amitet al., Science, 233:747-753 (1986)); interact with/effect theconformation of a CDR (Chothia et al., J. Mol. Biol., 196:901-917(1987)); and/or participate in the VL-VH interface (U.S. Pat. No.5,225,539).

In another embodiment the FR may comprise one or more amino acid changesfor the purposes of “germlining” For example, the amino acid sequencesof selected antibody heavy and light chains are compared to germlineheavy and light chain amino acid sequences and where certain frameworkresidues of the selected VL and/or VH chains differ from the germlineconfiguration (e.g., as a result of somatic mutation of theimmunoglobulin genes used to prepare the phage library), it may bedesirable to “backmutate” the altered framework residues of the selectedantibodies to the germline configuration (i.e., change the frameworkamino acid sequences of the selected antibodies so that they are thesame as the germline framework amino acid sequences). Such“backmutation” (or “germlining”) of framework residues can beaccomplished by standard molecular biology methods for introducingspecific mutations (e.g., site-directed mutagenesis; PCR-mediatedmutagenesis, and the like). In one embodiment, the variable light and/orheavy chain framework residues are backmutated. In another embodiment,the variable heavy chain of an antibody of the invention is backmutated.In another embodiment, the variable heavy chain of an antibody of theinvention comprises at least one, at least two, at least three, at leastfour or more backmutations.

In certain embodiments, the VH of an anti-TNFα antibody of the inventionmay comprise a FR1, FR2, FR3 and/or FR4 that has an amino acid sequenceidentity with the corresponding framework regions (i.e., FR1 of antibodyX as compared to FR1 of antibody Y) of any one or more of the VH chainsof the anti-TNFα antibodies described herein and set forth in theSequence Listing, that is from about 65% to about 100%. In oneembodiment, the anti-TNFα antibodies comprise, or have, a VH FR aminoacid sequence (e.g., FR1, FR2, FR3 and/or FR4) having at least 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with the corresponding FR of the VH set forth in SEQID NO:2.

In certain embodiments, the anti-TNFα antibodies containing a PGPKmodification may comprise a VH FR (e.g., FR1, FR2, FR3 and/or FR4)having an amino acid sequence identical to, or comprising 1, 2 or 3amino acid substitutions relative to, the corresponding FR of the VH setforth in SEQ ID NO:2.

In certain embodiments, the VL of an anti-TNFα antibody containing aPGPK modification of the invention may comprise a FR1, FR2, FR3 and/orFR4 that has an amino acid sequence identity with the correspondingframework regions (i.e., FR1 of antibody X as compared to FR1 ofantibody Y) of any one or more of the VH chains of the anti-TNFαantibodies described herein and set forth in the Sequence Listing, thatis from about 65% to about 100%. In one embodiment, the anti-TNFαantibodies containing a PGPK modification may comprise a VL FR aminoacid sequence (e.g., FR1, FR2, FR3 and/or FR4) having at least 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity with the corresponding FR of the VL chains set forthin SEQ ID NO:1.

5. Nucleotide Sequences Encoding Antibodies

In addition to the amino acid sequences described above, the presentinvention further provides nucleotide sequences corresponding to theamino acid sequences and encoding the antibodies of the invention. Inone embodiment, the present invention provides polynucleotidescomprising a nucleotide sequence encoding an anti-TNFα antibodycontaining a PGPK modification described herein or fragments thereof.These include, but are not limited to, nucleotide sequences that codefor the above referenced amino acid sequences. Thus, the presentinvention provides polynucleotide sequences encoding VH and VL domainregions including CDRs and FRs of antibodies described herein as well asexpression vectors for their efficient expression in cells (e.g.,mammalian cells). Methods of making the anti-TNFα antibodies usingpolynucleotides are described below in more detail and are known in theart. The foregoing polynucleotides encode anti-TNFα antibodiescontaining a PGPK modification and having the structural and/orfunctional features described herein.

The present invention also encompasses polynucleotides that hybridizeunder stringent hybridization conditions, e.g., as defined herein, topolynucleotides that encode an antibody of the invention.

Stringent hybridization conditions include, but are not limited to,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDSat about 50-65° C., highly stringent conditions such as hybridization tofilter-bound DNA in 6×SSC at about 45° C. followed by one or more washesin 0.1×SSC/0.2% SDS at about 65° C., or any other stringenthybridization conditions known to those skilled in the art (see, forexample, Ausubel, F. M. et al., eds. 1989 Current Protocols in MolecularBiology, vol. 1, Green Publishing Associates, Inc. and John Wiley andSons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3).

In certain embodiments, the polynucleotide sequences of the inventionmay also comprise a nucleotide sequence encoding an anti-TNFα antibodycontaining a PGPK modification VH which has at least 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to thenucleic acid sequence encoding SEQ ID NO:2.

In certain embodiments, the polynucleotide sequences of the inventionmay also comprise a nucleotide sequence encoding an anti-TNFα antibodycontaining a PGPK modification VL which has at least 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to thenucleotide sequence encoding SEQ ID NO:1.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

A polynucleotide encoding an antibody may also be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the immunoglobulinmay be chemically synthesized or obtained from a suitable source (e.g.,an antibody cDNA library, or a cDNA library generated from, or nucleicacid, in one aspect polyA+RNA, isolated from, any tissue or cellsexpressing the antibody, such as hybridoma cells selected to express anantibody) by PCR amplification using synthetic primers hybridizable tothe 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

6. Biological Characteristics of the Anti-TNFα Antibodies

The anti-TNFα antibodies, or antigen-binding fragments thereof, of theinvention comprising a PGPK modification are characterized by, or mayhave one or more of the biological characteristics described herein. Asused herein, the term “biological characteristics” of an antibody refersto any one of the biochemical, binding and functional characteristics,which are used to select antibodies for therapeutic, research, anddiagnostic uses as described, for example, in U.S. Pat. Nos. 6,090,382;6,258,562; 6,509,015; 7,223,394; 7,541,031; 7,588,761; 7,863,426;7,919,264; 8,197,813; 8,206,714; 8,216,583; 8,420,081; 8,092,998;8,093,045; 8,187,836; 8,372,400; 8,034,906; 8,436,149; 8,231,876;8,414,894; 8,372,401, PCT Publication No. WO2012/065072, the entirecontents of each which are expressly incorporated herein by reference.Such characteristics are also described, for example, in “Highlights ofPrescribing Information” for HUMIRA® (adalimumab) Injection (RevisedJanuary 2008); and below.

The biochemical characteristics of the antibodies of the inventioninclude, but are not limited to, isoelectric point (pI) and meltingtemperature (Tm). The binding characteristics of the antibodies of theinvention include, but are not limited to, binding specificity,dissociation constant (Kd), or its inverse, association constant (Ka),or its component k_(on) or k_(off) rates, epitope, ability todistinguish between various forms and/or preparations of TNFα (e.g.,recombinant, native, acetylated) and ability to bind soluble and/orimmobilized antigen. Methods for measuring the characteristics of theantibodies are well known in the art, some of which are detailed belowand in the examples section.

a. Biochemical Characteristics

Antibodies like all polypeptides have an Isoelectric Point (pI), whichis generally defined as the pH at which a polypeptide carries no netcharge. It is known in the art that protein solubility is typicallylowest when the pH of the solution is equal to the isoelectric point(pI) of the protein. As used herein the pI value is defined as the pI ofthe predominant charge form. The pI of a protein may be determined by avariety of methods including but not limited to, isoelectric focusingand various computer algorithms (see, e.g., Bjellqvist et al., 1993,Electrophoresis 14:1023). In addition, the thermal melting temperatures(Tm) of the Fab domain of an antibody, can be a good indicator of thethermal stability of an antibody and may further provide an indicationof the shelf-life. A lower Tm indicates more aggregation/less stability,whereas a higher Tm indicates less aggregation/more stability. Thus, incertain embodiments antibodies having higher Tm are desirable. Tm of aprotein domain (e.g., a Fab domain) can be measured using any standardmethod known in the art, for example, by differential scanningcalorimetry (see, e.g., Vermeer et al., 2000, Biophys. J. 78:394-404;Vermeer et al., 2000, Biophys. J. 79: 2150-2154).

Accordingly, in certain embodiments the present invention includesanti-TNFα antibodies containing a PGPK modification that have certaindesirable biochemical characteristics such as a particular isoelectricpoint (pI) or melting temperature (Tm).

More specifically, in one embodiment, the anti-TNFα antibodies of thepresent invention containing a PGPK modification may have a pI rangingfrom 5.5 to 9.5, e.g., about 5.5 to about 6.0, or about 6.0 to about6.5, or about 6.5 to about 7.0, or about 7.0 to about 7.5, or about 7.5to about 8.0, or about 8.0 to about 8.5, or about 8.5 to about 9.0, orabout 9.0 to about 9.5. In other specific embodiments, the anti-TNFαantibodies of the present invention containing a PGPK modification mayhave a pI that ranges from 5.5-6.0, or 6.0 to 6.5, or 6.5 to 7.0, or7.0-7.5, or 7.5-8.0, or 8.0-8.5, or 8.5-9.0, or 9.0-9.5. Even morespecifically, the anti-TNFα antibodies of the present inventioncontaining a PGPK modification may have a pI of at least 5.5, or atleast 6.0, or at least 6.3, or at least 6.5, or at least 6.7, or atleast 6.9, or at least 7.1, or at least 7.3, or at least 7.5, or atleast 7.7, or at least 7.9, or at least 8.1, or at least 8.3, or atleast 8.5, or at least 8.7, or at least 8.9, or at least 9.1, or atleast 9.3, or at least 9.5. In other specific embodiments, the anti-TNFαantibodies of the present invention containing a PGPK modification mayhave a pI of at least about 5.5, or at least about 6.0, or at leastabout 6.3, or at least about 6.5, or at least about 6.7, or at leastabout 6.9, or at least about 7.1, or at least about 7.3, or at leastabout 7.5, or at least about 7.7, or at least about 7.9, or at leastabout 8.1, or at least about 8.3, or at least about 8.5, or at leastabout 8.7, or at least about 8.9, or at least about 9.1, or at leastabout 9.3, or at least about 9.5.

It is possible to optimize solubility by altering the number andlocation of ionizable residues in the antibody to adjust the pI. Forexample the pI of a polypeptide can be manipulated by making theappropriate amino acid substitutions (e.g., by substituting a chargedamino acid such as a lysine, for an uncharged residue such as alanine).Without wishing to be bound by any particular theory, amino acidsubstitutions of an antibody that result in changes of the pI of theantibody may improve solubility and/or the stability of the antibody.One skilled in the art would understand which amino acid substitutionswould be most appropriate for a particular antibody to achieve a desiredpI. In one embodiment, a substitution is generated in an antibody of theinvention to alter the pI. It is specifically contemplated that thesubstitution(s) of the Fc region that result in altered binding to FcγR(described supra) may also result in a change in the pI. In anotherembodiment, substitution(s) of the Fc region are specifically chosen toeffect both the desired alteration in FcγR binding and any desiredchange in pI.

In one embodiment, the anti-TNFα antibodies of the present inventioncontaining a PGPK modification may have a Tm ranging from 65° C. to 120°C. In specific embodiments, the anti-TNFα antibodies of the presentinvention containing a PGPK modification may have a Tm ranging fromabout 75° C. to about 120° C., or about 75° C. to about 85° C., or about85° C. to about 95° C., or about 95° C. to about 105° C., or about 105°C. to about 115° C., or about 115° C. to about 120° C. In other specificembodiments, the anti-TNFα antibodies of the present inventioncontaining a PGPK modification may have a Tm ranging from 75° C. to 120°C., or 75° C. to 85° C., or 85° C. to 95° C., or 95° C. to 105° C., or105° C. to 115° C., or 115° C. to 120° C. In still other specificembodiments, the anti-TNFα antibodies of the present inventioncontaining a PGPK modification may have a Tm of at least about 65° C.,or at least about 70° C., or at least about 75° C., or at least about80° C., or at least about 85° C., or at least about 90° C., or at leastabout 95° C., or at least about 100° C., or at least about 105° C., orat least about 110° C., or at least about 115° C., or at least about120° C. In yet other specific embodiments, the anti-TNFα antibodies ofthe present invention containing a PGPK modification may have a Tm of atleast 65° C., or at least 70° C., or at least 75° C., or at least 80°C., or at least 85° C., or at least 90° C., or at least 95° C., or atleast 100° C., or at least 105° C., or at least 110° C., or at least115° C., or at least 120° C.

b. Binding Characteristics

As described above, the anti-TNFα antibodies of the invention containinga PGPK modification bind at least one epitope or antigenic determinantof TNFα or fragment thereof either exclusively or preferentially withrespect to other polypeptides. The term “epitope” is defined above, andincludes a protein determinant capable of binding to an antibody. Theseprotein determinants or epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnon-conformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents. The term “discontinuous epitope” as used herein, refers to aconformational epitope on a protein antigen which is formed from atleast two separate regions in the primary sequence of the protein. Incertain embodiments, the antibodies of the invention bind to human TNFαand antigenic fragments thereof. In certain embodiments, the anti-TNFαantibodies bind the same epitope as an antibody comprising the six CDRsof any of the antibodies listed in the Examples or Sequence Listing.

The antibodies of the invention may bind epitopes conserved acrossspecies. For example, antibodies of the invention may bind murine,non-human primate, rat, bovine, pig or other mammalian TNFα andantigenic fragments thereof. In one embodiment, the antibodies of theinvention may bind to one or more TNFα orthologs and or isoforms. In aspecific embodiment, antibodies of the invention may bind to TNFα andantigenic fragments thereof from one or more species, including, but notlimited to, mouse, rat, monkey, primate, and human. In certainembodiments, the antibodies of the invention may bind an epitope withinhumans across TNFα homologs and/or isoforms and/or conformationalvariants and/or subtypes.

The interactions between antigens and antibodies are the same as forother non-covalent protein-protein interactions. In general, four typesof binding interactions exist between antigens and antibodies: (i)hydrogen bonds, (ii) dispersion forces, (iii) electrostatic forcesbetween Lewis acids and Lewis bases, and (iv) hydrophobic interactions.Hydrophobic interactions are a major driving force for theantibody-antigen interaction, and are based on repulsion of water bynon-polar groups rather than attraction of molecules (Tanford, 1978).However, certain physical forces also contribute to antigen-antibodybinding, for example, the fit or complimentary of epitope shapes withdifferent antibody binding sites. Moreover, other materials and antigensmay cross-react with an antibody, thereby competing for available freeantibody.

Measurement of the affinity constant and specificity of binding betweenantigen and antibody is a pivotal element in determining the efficacy oftherapeutic, diagnostic and research methods using the anti-TNFαantibodies. “Binding affinity” generally refers to the strength of thesum total of the noncovalent interactions between a single binding siteof a molecule (e.g., an antibody) and its binding partner (e.g., anantigen). Unless indicated otherwise, as used herein, “binding affinity”refers to intrinsic binding affinity which reflects a 1:1 interactionbetween members of a binding pair (e.g., antibody and antigen). Theaffinity of a molecule X for its partner Y can generally be representedby the equilibrium dissociation constant (Kd), which is calculated asthe ratio k_(off)/k_(on). See, e.g., Chen, Y., et al., (1999) J. Mol.Biol 293:865-881. Affinity can be measured by common methods known inthe art, including those described and exemplified herein, such asBIACORE™. Low-affinity antibodies generally bind antigen slowly and tendto dissociate readily, whereas high-affinity antibodies generally bindantigen faster and tend to remain bound longer.

The anti-TNFα antibodies of the present invention containing a PGPKmodification may have binding affinities for a TNFα epitope that includea dissociation constant (K_(d)) of less than 1×10⁻²M, 1×10⁻³M, 1×10⁻⁴M,1×10⁻⁵M, 1×10⁻⁶M, 1×10⁻⁷M, 1×10⁻⁸M, 1×10⁻⁹M, 1×10⁻¹⁰M, 1×10⁻¹¹M,1×10⁻¹²M, 1×10⁻¹³M, 1×10⁻¹⁴M or less than 1×10⁻¹⁵M. In one embodiment,the anti-TNFα antibodies containing a PGPK modification may have a K_(d)of less than 10⁻⁷M, less than 5×10⁻⁸M, less than 10⁻⁸M, less than5×10⁻⁹M, less than 10⁻⁹M, less than 5×10⁻¹⁰M, less than 10⁻¹⁰M, lessthan 5×10⁻¹¹M, less than 10⁻¹¹M, less than 5×10⁻¹²M, less than 10⁻¹²M,less than 5×10⁻¹³M, less than 10⁻¹³M, less than 5×10⁻¹⁴M, less than10⁻¹⁴M, less than 5×10⁻¹⁵M, or less than 10⁻¹⁵ M. In certainembodiments, anti-TNFα antibodies containing a PGPK modification mayhave binding affinities for a TNFα epitope that include a dissociationconstant (K_(d)) of between 1×10⁻⁶M and 1×10⁻¹⁰M, 1×10⁻⁶M and 1×10⁻¹¹M,1×10⁻⁶M and 1×10⁻¹²M, 1×10⁻⁶M and 1×10⁻¹³M, 1×10⁻⁶M and 1×10⁻¹⁵M,1×10⁻⁶M and 1×10⁻¹⁵M, 1×10⁻⁷M and 1×10⁻¹⁰M, 1×10⁻⁷M and 1×10⁻¹¹M,1×10⁻⁷M and 1×10⁻¹²M, 1×10⁻⁷M and 1×10⁻¹³M, 1×10⁻⁷M and 1×10⁻¹⁴M,1×10⁻⁷M and 1×10⁻¹⁵M, 1×10⁻⁸M and 1×10⁻¹⁰M, 1×10⁻⁸M and 1×10⁻¹¹M,1×10⁻⁸M and 1×10⁻¹²M, 1×10⁻⁸M and 1×10⁻¹³M, 1×10⁻⁸M and 1×10⁻¹⁴M,1×10⁻⁸M and 1×10⁻¹⁵M, 1×10⁻⁹M and 1×10⁻¹¹M, 1×10⁻⁹M and 1×10⁻¹¹M,1×10⁻⁹M and 1×10⁻¹²M, 1×10⁻⁹M and 1×10⁻¹³M, 1×10⁻⁹M and 1×10⁻¹⁴M and1×10⁻⁹M and 1×10⁻¹⁵M. In certain embodiments, K_(d) is measured byBIACORE™. In certain embodiments, K_(d) is measured by cell binding.

In certain embodiments, the anti-TNFα antibodies containing a PGPKmodification are high-affinity antibodies. As used herein, the term“high affinity”, when referring to an IgG type antibody, refers to anantibody having a K_(D) of 10⁻⁸ M or less, alternatively 10⁻⁹ M or lessand alternatively 10⁻¹⁰ M or less for an antigen. However, “highaffinity” binding can vary for other antibody isotypes. For example,“high affinity” binding for an IgM isotype refers to an antibody havinga K_(D) of 10⁻⁷ M or less, 10⁻⁸ M or less, or 10⁻⁹ M or less for anantigen.

In certain embodiments, the anti-TNFα antibodies containing a PGPKmodification may have an affinity between 5 pM and 200 pM for activehuman TNFα, as assessed by plasmon resonance. In certain embodiments,the affinity is approximately 5, 10, 15, 20, 25, 50, 60, 70, 75, 80, 90,100, etc. pM. In certain embodiments, the affinity is between about 5 pMand 50 pM. In certain embodiments, the affinity is between about 5 pMand 100 pM.

In certain embodiments, the anti-TNFα antibodies containing a PGPKmodification are described as having a binding affinity of a specificmolarity or better. “Or better” when used herein refers to a strongerbinding, represented by a smaller numerical Kd value. For example, foran antibody which has an affinity for an antigen of “0.6 nM or better”,the antibody's affinity for the antigen is <0.6 nM, i.e., 0.59 nM, 0.58nM, 0.57 nM etc. or any value less than 0.6 nM.

In an alternative embodiment, the affinity of the anti-TNFα antibodiescontaining a PGPK modification is described in terms of the associationconstant (K_(a)), which is calculated as the ratio k_(on)/k_(off). Inthis instance the anti-TNFα antibodies containing a PGPK modificationmay have binding affinities for a TNFα epitope that include anassociation constant (K_(a)) of at least 1×10²M⁻¹, 1×10³M⁻¹, 1×10⁴M⁻¹,1×10⁵M⁻¹, 1×10⁶M⁻¹, 1×10⁷M⁻¹, 1×10⁸M⁻¹, 1×10⁹M⁻¹, 1×10¹⁰M⁻¹ 1×10¹¹M⁻¹1×10¹²M⁻¹, 1×10¹³M⁻¹, 1×10¹⁴M⁻¹ or at least, 1×10¹⁵M⁻¹. In oneembodiment, the anti-KIT antibodies have a K_(a) of at least 10⁷ M⁻¹, atleast 5×10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 5×10⁸ M⁻¹, at least 10⁹ M⁻¹,at least 5×10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 5×10¹⁰ M⁻¹, at least10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, at least 10¹² M⁻¹, at least 5×10¹² M⁻¹,at least 10¹³M⁻¹, at least 5×10¹³ M⁻¹, at least 10¹⁴ M⁻¹, at least5×10¹⁴ M⁻¹, at least 10¹⁵ M⁻¹, or at least 5×10¹⁵ M⁻¹. In certainembodiments, anti-TNFα antibodies containing a PGPK modification mayhave binding affinities for a TNFα epitope that include an associationconstant (K_(a)) of between 1×10²M⁻¹ and 1×10³M⁻¹, 1×10²M⁻¹ and1×10⁴M⁻¹, 1×10²M⁻¹ and 1×10⁵M⁻¹, 1×10²M⁻¹ and 1×10⁶M⁻¹, 1×10³M⁻¹ and1×10⁴M⁻¹, 1×10³M⁻¹ and 1×10⁵M⁻¹, 1×10³M⁻¹ and 1×10⁶M⁻¹, 1×10⁴M⁻¹ and1×10⁵M⁻¹, 1×10⁴M⁻¹ and 1×10⁶M⁻¹ and 1×10⁵M⁻¹ and 1×10⁶M⁻¹.

In certain embodiments the rate at which the anti-TNFα antibodiescontaining a PGPK modification may dissociate from a TNFα epitope may bemore relevant than the value of the K_(d) or the K_(a). In this instancethe anti-TNFα antibodies of the invention containing a PGPK modificationmay bind to TNFα, or a fragment thereof, with a k_(off) of less than10⁻² s⁻¹, less than 10⁻³ s⁻¹, less than 5×10⁻³ s⁻¹, less than 10⁻⁴ s⁻¹,less than 5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁵ s⁻¹, lessthan 10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁷ s⁻¹, less than5×10⁻⁷ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁹s⁻¹, less than 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹. In certain otherembodiments the rate at which the anti-TNFα antibodies containing a PGPKmodification may associate with a TNFα epitope may be more relevant thanthe value of the K_(d) or the K_(a). In this instance the anti-TNFαantibodies of the invention containing a PGPK modification may bind toTNFα, or a fragment thereof, with a k_(on) rate of at least 10⁵ M⁻¹ s⁻¹,at least 5×10⁵M⁻¹ s⁻¹, at least 10⁶M⁻¹ s⁻¹, at least 5×10⁶M⁻¹ s⁻¹, atleast 10⁷ M⁻¹ s⁻¹, at least 5×10⁷M⁻¹ s⁻¹, or at least 10⁸M⁻¹ s⁻¹, or atleast 10⁹M⁻¹ s⁻¹.

Determination of binding affinity can be measured using the specifictechniques described further in the Example section, and methods wellknown in the art. One such method includes measuring the disassociationconstant “Kd” by a radiolabeled antigen binding assay (RIA) performedwith the Fab version of an antibody of interest and its antigen asdescribed by the following assay that measures solution binding affinityof Fabs for antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol 293:865-881).To establish conditions for the assay, microtiter plates (Dynex) arecoated overnight with 5 μg/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (H 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbant plate (Nunc#269620), 100 pM 26 pM [¹²⁵I]-antigen are mixed with serial dilutions ofa Fab of interest (e.g., consistent with assessment of an anti-VEGFantibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599).The Fab of interest is then incubated overnight; however, the incubationmay continue for a longer period (e.g., 65 hours) to insure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 μl/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays.

The Kd value may also be measured by using surface plasmon resonanceassays using a BIACORE™-2000 or a BIACORE™-3000 (BIAcore, Inc.,Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at ^(˜)10response units (RU). Briefly, carboxymethylated dextran biosensor chips(CM5, BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 110 mM sodium acetate, pH 4.8, into 5 ug/ml(^(˜)0.2 uM) before injection at a flow rate of 5 ul/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, IM ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at25° C. at a flow rate of approximately 25 ul/min Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIACORE™ Evaluation Software version3.2) by simultaneously fitting the association and dissociationsensorgram.

If the on-rate exceeds 10⁶ M⁻¹S⁻¹ by the surface plasmon resonance assayabove, then the on-rate can be determined by using a fluorescentquenching technique that measures the increase or decrease influorescence emission intensity (excitation=295 nm; emission=340 nm, 16nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) inPBS, pH 7.2, in the presence of increasing concentrations of antigen asmeasured in a spectrometer, such as a stop-flow equipped spectrophometer(Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer(ThermoSpectronic) with a stir red cuvette. An “on-rate” or “rate ofassociation” or “association rate” or “k_(on)” according to thisinvention can also be determined with the same surface plasmon resonancetechnique described above using a BIACORE™-2000 or a BIACORE™-3000(BIAcore, Inc., Piscataway, N.J.) as described above.

Methods and reagents suitable for determination of bindingcharacteristics of an antibody of the present invention, or analtered/mutant derivative thereof (discussed below), are known in theart and/or are commercially available (see, for example, U.S. Pat. Nos.6,849,425; 6,632,926; 6,294,391; 6,143,574). Moreover, equipment andsoftware designed for such kinetic analyses are commercially available(e.g., BIACORE® A100, and BIACORE® 2000 instruments; BiacoreInternational AB, Uppsala, Sweden).

In one embodiment, a binding assay may be performed either as a directbinding assay or as a competition-binding assay. Binding can be detectedusing standard ELISA or standard Flow Cytometry assays. In a directbinding assay, a candidate antibody is tested for binding to TNFαantigen. Competition-binding assays, on the other hand, assess theability of a candidate antibody to compete with a known anti-TNFαantibody or other compound that binds TNFα. In general, any method thatpermits the detection and/or measuring of binding of an antibody withTNFα antigen may be used for detecting and measuring the bindingcharacteristics of the antibodies of the invention. One of skill in theart will recognize these well known methods and for this reason are notprovided in detail here.

c. Functional Characteristics

In certain embodiments, the antibodies of the invention containing aPGPK modification are anti-TNFα antibodies which modulate, e.g.,inhibit, human TNFα activity. In certain embodiments, the anti-TNFαantibodies of the instant invention will retain one or more specificanti-TNFα antibody activity. In certain embodiments, the anti-TNFαantibodies of the instant invention will retain activities such as thebinding specificity/affinity of an anti-TNFα antibody for its antigen,e.g., an anti-TNFα antibody that binds to a TNFα antigen and/or theneutralizing potency of an antibody, e.g., an anti-TNFα antibody whosebinding to hTNFα inhibits the biological activity of hTNFα.

In certain embodiments, the compositions of the present inventioninclude anti-TNFα antibodies that dissociate from hTNFα with a K_(d) ofabout 1×10⁻⁸ M or less and a K_(off) rate constant of 1×10⁻³ s⁻¹ orless, both determined, for example, but not by way of limitation, bysurface plasmon resonance. In specific non-limiting embodiments, theanti-TNFα antibodies of the instant invention competitively inhibit thebinding of adalimumab to TNFα under physiological conditions.

In one embodiment, the antibodies of the invention exhibit a reducedantibody related toxicity as compared to previously describedantibodies.

In another embodiment, the antibodies of the invention exhibit increasedtissue (e.g., cartilage) penetration, increased TNFα affinity, reducedcartilage destruction, reduced bone erosion, reduced synovialproliferation, reduced cell infiltration, reduced chondrocyte death,reduced proteoglycan loss, increased protection against the developmentof arthritis scores when administered to an animal model of arthritis,and/or increased protection against the development of histopathologyscores when administered to an animal model of arthritis as compared topreviously described antibodies, e.g., antibodies not containing a PGPKmodification.

In certain embodiments, the antibodies of the invention may bindepitopes conserved across species. In one embodiment, antibodies of theinvention bind murine, non-human primate, rat, bovine, pig or othermammalian TNFα and antigenic fragments thereof. In one embodiment theantibodies of the invention may bind to one or more TNFα orthologs andor isoforms. In a specific embodiment, antibodies of the invention bindto TNFα and antigenic fragments thereof from one or more species,including, but not limited to, mouse, rat, monkey, primate, and human.In certain embodiments, the antibodies of the invention may bind anepitope within humans across TNFα homologs and/or isoforms and/orconformational variants and/or subtypes.

II. Expression and Production of Antibodies

The following section describes exemplary techniques for the productionof the antibodies, and antigen-binding portions thereof, containing aPGPK modification of the invention. Such techniques are merelyexemplary.

To express an antibody, or antigen-binding portion thereof, comprising aPGPK modification of the invention, DNA(s) encoding the antibody, orantigen-binding portion thereof, such as DNA(s) encoding partial orfull-length light and heavy chains, may be inserted into one or moreexpression vector such that the genes are operatively linked totranscriptional and translational control sequences (see, e.g., U.S.Pat. Nos. 6,090,382; 6,258,562; 6,509,015; 7,223,394; 7,541,031;7,588,761; 7,863,426; 7,919,264; 8,197,813; 8,206,714; 8,216,583;8,420,081; 8,092,998; 8,093,045; 8,187,836; 8,372,400; 8,034,906;8,436,149; 8,231,876; 8,414,894; 8,372,401, and PCT Publication No.WO2012/065072, the entire contents of each which are expresslyincorporated herein by reference in their entireties). In this context,the term “operatively linked” is intended to mean that a gene encodingthe antibody, or antigen-binding portion thereof, of interest is ligatedinto a vector such that transcriptional and translational controlsequences within the vector serve their intended function of regulatingthe transcription and translation of the gene. The expression vector andexpression control sequences are chosen to be compatible with theexpression host cell used. In certain embodiments, the antibody, orantigen-binding portion thereof comprising a PGPK modification willcomprise multiple polypeptides, such as the heavy and light chains.Thus, in certain embodiments, genes encoding multiple polypeptides, suchas antibody light chain genes and antibody heavy chain genes, can beinserted into a separate vector or, more typically, the genes areinserted into the same expression vector. Genes are inserted intoexpression vectors by standard methods (e.g., ligation of complementaryrestriction sites on the gene fragment and vector, or blunt end ligationif no restriction sites are present). Prior to insertion of the gene orgenes, the expression vector may already carry additional polypeptidesequences, such as, but no limited to, antibody constant regionsequences. For example, one approach to converting an antibody orantibody-related VH and VL sequences to full-length antibody genes is toinsert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theVH segment is operatively linked to the CH segment(s) within the vectorand the VL segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the proteinfrom a host cell. The gene can be cloned into the vector such that thesignal peptide is linked in-frame to the amino terminus of the gene. Thesignal peptide can be an immunoglobulin signal peptide or a heterologoussignal peptide (i.e., a signal peptide from a non-immunoglobulinprotein).

In addition to protein coding genes, a recombinant expression vector ofthe invention can carry one or more regulatory sequence that controlsthe expression of the protein coding genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the protein coding genes.Such regulatory sequences are described, e.g., in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990), the entire teaching of which is incorporatedherein by reference. It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, see,e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al., the entirecontents of which are expressly incorporated herein by reference.

In addition to the protein coding genes and regulatory sequences, arecombinant expression vector of the invention may carry one or moreadditional sequences, such as a sequence that regulates replication ofthe vector in host cells (e.g., origins of replication) and/or aselectable marker gene. The selectable marker gene facilitates selectionof host cells into which the vector has been introduced (see e.g., U.S.Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al., theentire contents of which are incorporated herein by reference). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Suitable selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr-hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

An antibody, or antibody portion, comprising a PGPK modification of theinvention can be prepared by recombinant expression of immunoglobulinlight and heavy chain genes in a host cell. To express an antibodyrecombinantly, a host cell is transfected with one or more recombinantexpression vectors carrying DNA fragments encoding the immunoglobulinlight and heavy chains of the antibody such that the light and heavychains are expressed in the host cell and secreted into the medium inwhich the host cells are cultured, from which medium the antibodies canbe recovered. Standard recombinant DNA methodologies are used to obtainantibody heavy and light chain genes, incorporate these genes intorecombinant expression vectors and introduce the vectors into hostcells, such as those described in Sambrook, Fritsch and Maniatis (eds),Molecular Cloning; A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), Ausubel et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.Nos. 6,090,382; 6,258,562; 6,509,015; 7,223,394; 7,541,031; 7,588,761;7,863,426; 7,919,264; 8,197,813; 8,206,714; 8,216,583; 8,420,081;8,092,998; 8,093,045; 8,187,836; 8,372,400; 8,034,906; 8,436,149;8,231,876; 8,414,894; 8,372,401, the entire contents of each which areexpressly incorporated herein by reference in their entireties.

For expression of an antibody, or antigen-binding portion thereof,comprising a PGPK modification of the invention, for example, theexpression vector(s) encoding the protein is (are) transfected into ahost cell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it istheoretically possible to express the proteins of the invention ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, such as mammalian host cells, is suitable because sucheukaryotic cells, and in particular mammalian cells, are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active protein. Prokaryotic expression of protein geneshas been reported to be ineffective for production of high yields ofactive protein (Boss and Wood (1985) Immunology Today 6:12-13, theentire contents of which are incorporated herein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, e.g., Enterobacteriaceae suchas Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratia marcescans, and Shigella, as well as Bacilli such as B.subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed inDD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa,and Streptomyces. One suitable E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424),K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated proteins, forexample, glycosylated antibodies, are derived from multicellularorganisms. Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains and variants and correspondingpermissive insect host cells from hosts such as Spodoptera frugiperda(caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),Drosophila melanogaster (fruitfly), and Bombyx mori have beenidentified. A variety of viral strains for transfection are publiclyavailable, e.g., the L-1 variant of Autographa californica NPV and theBm-5 strain of Bombyx mori NPV, and such viruses may be used as thevirus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

Mammalian cells may be used for expression and production of therecombinant protein of the present invention, however other eukaryoticcell types can also be employed in the context of the instant invention.See, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y.(1987). Suitable mammalian host cells for expressing recombinantproteins according to the invention include Chinese Hamster Ovary (CHOcells) (including dhfr-CHO cells, described in Urlaub and ChasM, (1980)PNAS USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp (1982) Mol. Biol. 159:601-621, the entireteachings of which are incorporated herein by reference), NS0 myelomacells, COS cells and SP2 cells. When recombinant expression vectorsencoding protein genes are introduced into mammalian host cells, theantibodies are produced by culturing the host cells for a period of timesufficient to allow for expression of the antibody in the host cells orsecretion of the antibody into the culture medium in which the hostcells are grown. Other examples of useful mammalian host cell lines aremonkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2), the entire teachings of which are incorporated herein byreference.

Host cells are transformed with the above-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce a protein may be cultured in a variety ofmedia. Commercially available media such as Ham's F10™ (Sigma), MinimalEssential Medium™ (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium™ (DMEM), (Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham et al., Meth.Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used asculture media for the host cells, the entire teachings of which areincorporated herein by reference. Any of these media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asgentamycin drug), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH, andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

Host cells can also be used to produce portions of intact proteins, forexample, antibodies, including Fab fragments or scFv molecules. It isunderstood that variations on the above procedure are within the scopeof the present invention. For example, in certain embodiments it may bedesirable to transfect a host cell with DNA encoding either the lightchain or the heavy chain (but not both) of an antibody. Recombinant DNAtechnology may also be used to remove some or all of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to an antigen. The molecules expressed from such truncated DNAmolecules are also encompassed by the antibodies of the invention. Inaddition, bifunctional antibodies may be produced in which one heavy andone light chain are an antibody of the invention and the other heavy andlight chain are specific for an antigen other than the target antibody,depending on the specificity of the antibody of the invention, bycrosslinking an antibody of the invention to a second antibody bystandard chemical crosslinking methods.

In a suitable system for recombinant expression of a protein, forexample, an antibody, or antigen-binding portion thereof, a recombinantexpression vector encoding the protein, for example, both an antibodyheavy chain and an antibody light chain, is introduced into dhfr-CHOcells by calcium phosphate-mediated transfection. Within the recombinantexpression vector, the protein gene(s) are each operatively linked toCMV enhancer/AdMLP promoter regulatory elements to drive high levels oftranscription of the gene(s). The recombinant expression vector alsocarries a DHFR gene, which allows for selection of CHO cells that havebeen transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the protein, for example, theantibody heavy and light chains, and intact protein, for example, anantibody, is recovered from the culture medium. Standard molecularbiology techniques are used to prepare the recombinant expressionvector, transfect the host cells, select for transformants, culture thehost cells and recover the protein from the culture medium.

When using recombinant techniques, the protein, for example, antibodiesor antigen binding fragments thereof, can be produced intracellularly,in the periplasmic space, or directly secreted into the medium. In oneaspect, if the protein is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed cells (e.g., resultingfrom homogenization), can be removed, e.g., by centrifugation orultrafiltration. Where the protein is secreted into the medium,supernatants from such expression systems can be first concentratedusing a commercially available protein concentration filter, e.g., anAmicon™ or Millipore Pellicon™ ultrafiltration unit.

Some antibodies can be secreted directly from the cell into thesurrounding growth media; others are made intracellularly. Forantibodies made intracellularly, the first step of a purificationprocess typically involves: lysis of the cell, which can be done by avariety of methods, including mechanical shear, osmotic shock, orenzymatic treatments. Such disruption releases the entire contents ofthe cell into the homogenate, and in addition produces subcellularfragments that are difficult to remove due to their small size. Theseare generally removed by differential centrifugation or by filtration.Where the antibody is secreted, supernatants from such expressionsystems are generally first concentrated using a commercially availableprotein concentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit. Where the antibody is secreted into the medium,the recombinant host cells can also be separated from the cell culturemedium, e.g., by tangential flow filtration. Antibodies can be furtherrecovered from the culture medium using the antibody purificationmethods of the invention, described below.

Antibody Conjugates

The antibodies, or antigen-binding portions thereof, of the inventioncomprising a PGPK modification may be conjugated or covalently attachedto a substance using methods well known in the art. In one embodiment,the attached substance is a therapeutic agent, a detectable label (alsoreferred to herein as a reporter molecule) or a solid support. Suitablesubstances for attachment to antibodies include, but are not limited to,an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell, a virus, a fluorophore, a chromophore,a dye, a toxin, a hapten, an enzyme, an antibody, an antibody fragment,a radioisotope, solid matrixes, semi-solid matrixes and combinationsthereof. In a specific embodiment, the attached substance is a toxin. Inanother embodiment, the attached substance is an anti-cancer agent.Methods for conjugation or covalently attaching another substance to anantibody are well known in the art.

The antibodies of the invention may also be conjugated to a solidsupport. Antibodies may be conjugated to a solid support as part of thescreening and/or purification and/or manufacturing process.Alternatively antibodies of the invention may be conjugated to a solidsupport as part of a diagnostic method or composition. A solid supportsuitable for use in the present invention is typically substantiallyinsoluble in liquid phases. A large number of supports are available andare known to one of ordinary skill in the art. Thus, solid supportsinclude solid and semi-solid matrixes, such as aerogels and hydrogels,resins, beads, biochips (including thin film coated biochips),microfluidic chip, a silicon chip, multi-well plates (also referred toas microtitre plates or microplates), membranes, conducting andnonconducting metals, glass (including microscope slides) and magneticsupports. More specific examples of solid supports include silica gels,polymeric membranes, particles, derivatized plastic films, glass beads,cotton, plastic beads, alumina gels, polysaccharides such as Sepharose,poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride,polypropylene, polyethylene (including poly(ethylene glycol)), nylon,latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead,starch and the like.

In some embodiments, the solid support may include a reactive functionalgroup, including, but not limited to, hydroxyl, carboxyl, amino, thiol,aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate,isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, etc., forattaching the antibodies of the invention.

A suitable solid phase support can be selected on the basis of desiredend use and suitability for various synthetic protocols. For example,where amide bond formation is desirable to attach the antibodies of theinvention to the solid support, resins generally useful in peptidesynthesis may be employed, such as polystyrene (e.g., PAM-resin obtainedfrom Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin(obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TENTAGEL™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories).

The antibodies of the invention may also be conjugated to labels forpurposes of diagnostics and other assays wherein the antibody and/or itsassociated ligand may be detected. A label conjugated to an antibody andused in the present methods and compositions described herein, is anychemical moiety, organic or inorganic, that exhibits an absorptionmaximum at wavelengths greater than 280 nm, and retains its spectralproperties when covalently attached to an antibody. Labels include,without limitation, a chromophore, a fluorophore, a fluorescent protein,a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme anda radioisotope.

In certain embodiments, the antibodies of the invention are conjugatedto a fluorophore. As such, fluorophores used to label antibodies of theinvention include, without limitation; a pyrene (including any of thecorresponding derivative compounds disclosed in U.S. Pat. No.5,132,432), an anthracene, a naphthalene, an acridine, a stilbene, anindole or benzindole, an oxazole or benzoxazole, a thiazole orbenzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine(including any corresponding compounds in U.S. Pat. Nos. 6,977,305 and6,974,873), a carbocyanine (including any corresponding compounds inU.S. Ser. No. 09/557,275; U.S. Pat. Nos. 4,981,977; 5,268,486;5,569,587; 5,569,766; 5,486,616; 5,627,027; 5,808,044; 5,877,310;6,002,003; 6,004,536; 6,008,373; 6,043,025; 6,127,134; 6,130,094;6,133,445; and publications WO 02/26891, WO 97/40104, WO 99/51702, WO01/21624; EP 1 065 250 A1), a carbostyryl, a porphyrin, a salicylate, ananthranilate, an azulene, a perylene, a pyridine, a quinoline, aborapolyazaindacene (including any corresponding compounds disclosed inU.S. Pat. Nos. 4,774,339; 5,187,288; 5,248,782; 5,274,113; and5,433,896), a xanthene (including any corresponding compounds disclosedin U.S. Pat. Nos. 6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343;5,227,487; 5,442,045; 5,798,276; 5,846,737; 4,945,171; U.S. Ser. Nos.09/129,015 and 09/922,333), an oxazine (including any correspondingcompounds disclosed in U.S. Pat. No. 4,714,763) or a benzoxazine, acarbazine (including any corresponding compounds disclosed in U.S. Pat.No. 4,810,636), a phenalenone, a coumarin (including an correspondingcompounds disclosed in U.S. Pat. Nos. 5,696,157; 5,459,276; 5,501,980and 5,830,912), a benzofuran (including an corresponding compoundsdisclosed in U.S. Pat. Nos. 4,603,209 and 4,849,362) and benzphenalenone(including any corresponding compounds disclosed in U.S. Pat. No.4,812,409) and derivatives thereof. As used herein, oxazines includeresorufins (including any corresponding compounds disclosed in U.S. Pat.No. 5,242,805), aminooxazinones, diaminooxazines, and theirbenzo-substituted analogs.

In a specific embodiment, the fluorophores conjugated to the antibodiesdescribed herein include xanthene (rhodol, rhodamine, fluorescein andderivatives thereof) coumarin, cyanine, pyrene, oxazine andborapolyazaindacene. In other embodiments, such fluorophores aresulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins,fluorinated coumarins and sulfonated cyanines. Also included are dyessold under the tradenames, and generally known as, Alexa Fluor, DyLight,Cy Dyes, BODIPY, Oregon Green, Pacific Blue, IRDyes, FAM, FITC, and ROX.

The choice of the fluorophore attached to the antibody will determinethe absorption and fluorescence emission properties of the conjugatedantibody. Physical properties of a fluorophore label that can be usedfor antibody and antibody bound ligands include, but are not limited to,spectral characteristics (absorption, emission and stokes shift),fluorescence intensity, lifetime, polarization and photo-bleaching rate,or combination thereof. All of these physical properties can be used todistinguish one fluorophore from another, and thereby allow formultiplexed analysis. In certain embodiments, the fluorophore has anabsorption maximum at wavelengths greater than 480 nm. In otherembodiments, the fluorophore absorbs at or near 488 nm to 514 nm(particularly suitable for excitation by the output of the argon-ionlaser excitation source) or near 546 nm (particularly suitable forexcitation by a mercury arc lamp). In other embodiment a fluorophore canemit in the NIR (near infra red region) for tissue or whole organismapplications. Other desirable properties of the fluorescent label mayinclude cell permeability and low toxicity, for example if labeling ofthe antibody is to be performed in a cell or an organism (e.g., a livinganimal).

In certain embodiments, an enzyme is a label and is conjugated to anantibody of the invention. Enzymes are desirable labels becauseamplification of the detectable signal can be obtained resulting inincreased assay sensitivity. The enzyme itself does not produce adetectable response but functions to break down a substrate when it iscontacted by an appropriate substrate such that the converted substrateproduces a fluorescent, colorimetric or luminescent signal. Enzymesamplify the detectable signal because one enzyme on a labeling reagentcan result in multiple substrates being converted to a detectablesignal. The enzyme substrate is selected to yield the measurableproduct, e.g., colorimetric, fluorescent or chemiluminescence. Suchsubstrates are extensively used in the art and are well known by oneskilled in the art.

In one embodiment, colorimetric or fluorogenic substrate and enzymecombination uses oxidoreductases such as horseradish peroxidase and asubstrate such as 3,3′-diaminobenzidine (DAB) and3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color(brown and red, respectively). Other colorimetric oxidoreductasesubstrates that yield detectable products include, but are not limitedto: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. Fluorogenicsubstrates include, but are not limited to, homovanillic acid or4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reducedbenzothiazines, including AMPLEX® Red reagent and its variants (U.S.Pat. No. 4,384,042) and reduced dihydroxanthenes, includingdihydrofluoresceins (U.S. Pat. No. 6,162,931) and dihydrorhodaminesincluding dihydrorhodamine 123. Peroxidase substrates that are tyramides(U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158) represent a uniqueclass of peroxidase substrates in that they can be intrinsicallydetectable before action of the enzyme but are “fixed in place” by theaction of a peroxidase in the process described as tyramide signalamplification (TSA). These substrates are extensively utilized to labeltargets in samples that are cells, tissues or arrays for theirsubsequent detection by microscopy, flow cytometry, optical scanning andfluorometry.

In another embodiment, a colorimetric (and in some cases fluorogenic)substrate and enzyme combination uses a phosphatase enzyme such as anacid phosphatase, an alkaline phosphatase or a recombinant version ofsuch a phosphatase in combination with a colorimetric substrate such as5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolylphosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenylphosphate, or o-nitrophenyl phosphate or with a fluorogenic substratesuch as 4-methylumbelliferyl phosphate,6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.No. 5,830,912) fluorescein diphosphate, 3-O-methylfluorescein phosphate,resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphates(U.S. Pat. Nos. 5,316,906 and 5,443,986).

Glycosidases, in particular beta-galactosidase, beta-glucuronidase andbeta-glucosidase, are additional suitable enzymes. Appropriatecolorimetric substrates include, but are not limited to,5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similarindolyl galactosides, glucosides, and glucuronides, o-nitrophenylbeta-D-galactopyranoside (ONPG) and p-nitrophenylbeta-D-galactopyranoside. In one embodiment, fluorogenic substratesinclude resorufin beta-D-galactopyranoside, fluorescein digalactoside(FDG), fluorescein diglucuronide and their structural variants (U.S.Pat. Nos. 5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236),4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferylbeta-D-galactopyranoside and fluorinated coumarinbeta-D-galactopyranosides (U.S. Pat. No. 5,830,912).

Additional enzymes include, but are not limited to, hydrolases such ascholinesterases and peptidases, oxidases such as glucose oxidase andcytochrome oxidases, and reductases for which suitable substrates areknown.

Enzymes and their appropriate substrates that produce chemiluminescenceare desirable for some assays. These include, but are not limited to,natural and recombinant forms of luciferases and aequorins.Chemiluminescence-producing substrates for phosphatases, glycosidasesand oxidases such as those containing stable dioxetanes, luminol,isoluminol and acridinium esters are additionally useful.

In another embodiment, haptens such as biotin, are also utilized aslabels. Biotin is useful because it can function in an enzyme system tofurther amplify the detectable signal, and it can function as a tag tobe used in affinity chromatography for isolation purposes. For detectionpurposes, an enzyme conjugate that has affinity for biotin is used, suchas avidin-HRP. Subsequently a peroxidase substrate is added to produce adetectable signal.

Haptens also include hormones, naturally occurring and synthetic drugs,pollutants, allergens, affector molecules, growth factors, chemokines,cytokines, lymphokines, amino acids, peptides, chemical intermediates,nucleotides and the like.

In certain embodiments, fluorescent proteins may be conjugated to theantibodies as a label. Examples of fluorescent proteins include greenfluorescent protein (GFP) and the phycobiliproteins and the derivativesthereof. The fluorescent proteins, especially phycobiliprotein, areparticularly useful for creating tandem dye labeled labeling reagents.These tandem dyes comprise a fluorescent protein and a fluorophore forthe purposes of obtaining a larger stokes shift wherein the emissionspectra is farther shifted from the wavelength of the fluorescentprotein's absorption spectra. This is particularly advantageous fordetecting a low quantity of a target in a sample wherein the emittedfluorescent light is maximally optimized, in other words little to noneof the emitted light is reabsorbed by the fluorescent protein. For thisto work, the fluorescent protein and fluorophore function as an energytransfer pair wherein the fluorescent protein emits at the wavelengththat the fluorophore absorbs at and the fluorophore then emits at awavelength farther from the fluorescent proteins than could have beenobtained with only the fluorescent protein. A particularly usefulcombination is the phycobiliproteins disclosed in U.S. Pat. Nos.4,520,110; 4,859,582; 5,055,556 and the sulforhodamine fluorophoresdisclosed in U.S. Pat. No. 5,798,276, or the sulfonated cyaninefluorophores disclosed in U.S. Pat. Nos. 6,977,305 and 6,974,873; or thesulfonated xanthene derivatives disclosed in U.S. Pat. No. 6,130,101 andthose combinations disclosed in U.S. Pat. No. 4,542,104. Alternatively,the fluorophore functions as the energy donor and the fluorescentprotein is the energy acceptor.

In certain embodiments, the label is a radioactive isotope. Examples ofsuitable radioactive materials include, but are not limited to, iodine(¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹¹¹In, ¹¹²In, ¹¹³mIn, ¹¹⁵mIn), technetium (⁹⁹Tc, ⁹⁹mTc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³⁵Xe), fluorine (¹⁸F), ¹⁵³SM, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh and ⁹⁷ Ru.

III. Antibody Purification and Isolation

Once an antibody of the invention, e.g., an anti-TNFα antibodycomprising a PGPK modification, has been expressed or produced, it maybe purified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigensProtein A or Protein G, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins. In certain embodiments, theinvention provides methods and compositions for producing a purified orpartially purified (e.g., process-related impurity-reduced and/orproduct-related substance-reduced) protein preparation from a mixturecomprising a protein of interest, e.g., an anti-TNFα antibody, orantigen-binding portion thereof, and at least one process-relatedimpurity or product-related substance. In certain embodiments, thecompositions of the present invention include, but are not limited to,process-related impurity-reduced and/or product-relatedsubstance-reduced compositions comprising a protein of interest. Suchprocess-related impurity-reduced and/or product-relatedsubstance-reduced compositions address the need for improved productcharacteristics, including, but not limited to, product stability,product safety and product efficacy.

In certain embodiments, the purification process of the invention beginsat the separation step when the antibody has been produced usingproduction methods described above and/or by alternative productionmethods conventional in the art. Once a clarified solution or mixturecomprising the protein of interest, e.g., an antibody, has beenobtained, separation of the protein of interest from process-relatedimpurities, such as the other proteins produced by the cell, as well asany product-related substances such as charge variants and/or sizevariants (aggregates and fragments), can be performed. In certainnon-limiting embodiments, such separation is performed using Protein Aaffinity chromatography followed by a displacement chromatographic step.Such chromatographic steps may include, but are not limited to, ionexchange, mixed mode exchange, hydrophobic interaction chromatography,etc., as described herein and in U.S. Provisional Patent Application61/893,068, entitled “Low Acidic Species Compositions and Methods forProducing and Using the Same,” filed on Oct. 18, 2013, the entirecontents of which are expressly incorporated herein by reference. Incertain embodiments, a combination of one or more different purificationtechniques described herein, including, but not limited to, ion exchangeseparation step(s) and/or hydrophobic interaction separation step(s) canalso be employed. Such additional purification steps separate mixturesof proteins on the basis of their charge, degree of hydrophobicity,and/or size. In one aspect of the invention, such additional separationsteps are performed using chromatography, including hydrophobic, anionicor cationic, or mixed mode interaction. Several different chromatographyresins are available for each of these techniques, allowing accuratetailoring of the purification scheme to the particular protein involved.The essence of each of the separation methods is that proteins caneither traverse at different rates through a column, achieving aphysical separation that increases as they pass further down the column,or to adhere selectively to the separation medium, being thendifferentially eluted by different solvents, or by the presence of adisplacer (in the context of displacement chromatography). In somecases, the antibody is separated from impurities when the impuritiesspecifically adhere to the column and the antibody does not, i.e., theantibody is present in the flow-through, while in other cases theantibody will adhere to the column, while the impurities flow-through.

1. Primary Recovery

In certain embodiments, the initial steps of the purification methods ofthe present invention involve the clarification and primary recovery ofantibody from a sample matrix. In certain embodiments, the primaryrecovery will include one or more centrifugation steps to separate theantibody product from the cells and cell debris. Centrifugation of thesample can be run at, for example, but not by way of limitation, 7,000×gto approximately 12,750×g. In the context of large scale purification,such centrifugation can occur on-line with a flow rate set to achieve,for example, but not by way of limitation, a turbidity level of 150 NTUin the resulting supernatant. Such supernatant can then be collected forfurther purification, or in-line filtered through one or more depthfilters for further clarification of the sample.

In certain embodiments, the primary recovery will include the use of oneor more depth filtration steps to clarify the sample matrix and therebyaid in purifying the antibodies of interest in the present invention. Inother embodiments, the primary recovery will include the use of one ormore depth filtration steps post centrifugation to further clarify thesample matrix. Non-limiting examples of depth filters that can be usedin the context of the instant invention include the Millistak+X0HC,F0HC, D0HC, A1HC, B1HC depth filters (EMD Millipore), Cuno™ model30/60ZA, 60/90 ZA, VR05, VR07, delipid depth filters (3M Corp.). A 0.2μm filter such as Sartorius's 0.45/0.2 μm Sartopore™ bi-layer orMillipore's Express SHR or SHC filter cartridges typically follows thedepth filters.

In certain embodiments, the primary recovery process can also be a pointat which to reduce or inactivate viruses that can be present in thesample matrix. For example, any one or more of a variety of methods ofviral reduction/inactivation can be used during the primary recoveryphase of purification including heat inactivation (pasteurization), pHinactivation, solvent/detergent treatment, UV and γ-ray irradiation andthe addition of certain chemical inactivating agents such asβ-propiolactone or e.g., copper phenanthroline as in U.S. Pat. No.4,534,972. In certain embodiments of the present invention, the samplematrix is exposed to detergent viral inactivation during the primaryrecovery phase. In other embodiments, the sample matrix may be exposedto low pH inactivation during the primary recovery phase.

In those embodiments where viral reduction/inactivation is employed, thesample mixture can be adjusted, as needed, for further purificationsteps. For example, following low pH viral inactivation, the pH of thesample mixture is typically adjusted to a more neutral pH, e.g., fromabout 4.5 to about 8.5, prior to continuing the purification process.Additionally, the mixture may be diluted with water for injection (WFI)to obtain a desired conductivity.

2. Protein A Affinity Chromatography

In certain embodiments, the primary recovery sample is subjected toProtein A affinity chromatography to substantially purify the antibodyof interest away from host cell proteins (“HCPs”). There are a varietyof commercial sources for Protein A resin. Suitable resins include, butnot limited to, MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelectXtra, rProtein A Sepharose from GE Healthcare, ProSep HC, ProSep Ultra,and ProSep Ultra Plus from EMD Millipore, MapCapture from LifeTechnologies.

In certain embodiments, the Protein A column can be equilibrated with asuitable buffer prior to sample loading. Following the loading of thecolumn, the column can be washed one or multiple times using a suitablesets of buffers. The Protein A column can then be eluted using anappropriate elution buffer. The eluate can be monitored using techniqueswell known to those skilled in the art. The eluate fractions of interestcan be collected and then prepared for further processing.

The Protein A eluate may subject to a viral inactivation step either bydetergent or low pH, provided this step is not performed prior to theProtein A capture operation. A proper detergent concentration or pH andtime can be selected to obtain desired viral inactivation results. Afterviral inactivation, the Protein A eluate is usually pH and/orconductivity adjusted for subsequent purification steps.

The Protein A eluate may be subjected to filtration through a depthfilter to remove turbidity and/or various impurities from the antibodyof interest prior to additional chromatographic polishing steps.Examples of depth filters include, but not limited to, Millistak+X0HC,F0HC, D0HC, A1HC, and B1HC Pod filters (EMD Millipore), or Zeta Plus30ZA/60ZA, 60ZA/90ZA, delipid, VR07, and VR05 filters (3M). The ProteinA eluate pool may need to be conditioned to proper pH and conductivityto obtain desired impurity removal and product recovery from the depthfiltration step.

3. Ion Exchange Chromatography

In certain embodiments, an ion exchange step will occur after theabove-described Protein A affinity and/or displacement chromatographysteps, such that an eluate comprising the protein of interest isobtained. Ion exchange separation includes any method by which twosubstances are separated based on the difference in their respectiveionic charges, and can employ either cationic exchange material oranionic exchange material.

The use of a cationic exchange material versus an anionic exchangematerial can be based on the local charges of the protein at a givensolution condition. Therefore, it is within the scope of this inventionto employ an anionic exchange step prior to or subsequent to the use ofa displacement chromatography step, or a cationic exchange step prior toor subsequent to the use of a displacement chromatography step.

In performing the separation, the initial protein mixture can becontacted with the ion exchange material by using any of a variety oftechniques, e.g., using a batch purification technique or achromatographic technique.

For example, in the context of batch purification, ion exchange materialis prepared in, or equilibrated to, the desired starting buffer. Uponpreparation, or equilibration, a slurry of the ion exchange material isobtained. The protein of interest, e.g., an antibody, solution iscontacted with the slurry to adsorb the protein of interest to beseparated to the ion exchange material. The solution comprising theprocess-related impurities and product-related substances that do notbind to the ion exchange material is separated from the slurry, e.g., byallowing the slurry to settle and removing the supernatant. The slurrycan be subjected to one or more wash steps. If desired, the slurry canbe contacted with a solution of higher conductivity to desorbprocess-related impurities and product-related substances that havebound to the ion exchange material. In order to elute boundpolypeptides, the salt concentration of the buffer can be increased.

In the context of chromatographic separation, a chromatographicapparatus, commonly cylindrical in shape, is employed to contain thechromatographic support material (e.g., ion exchange material) preparedin an appropriate buffer solution. The chromatographic apparatus, ifcylindrical, can have a diameter of about 5 mm to about 50 mm, and aheight of 5 cm to 1 m, and in certain embodiments, particularly forlarge scale processing, a height of <30 cm is employed. Once thechromatographic material is added to the chromatographic apparatus, asample containing the protein of interest, e.g., an antibody, iscontacted to the chromatographic material to adsorb the protein ofinterest to be separated to the chromatographic material. The solutioncomprising the process-related impurities and product-related substancesthat do not bind to the chromatographic material is separated from thematerial by washing the materials and collecting fractions from thebottom of the column. The chromatographic material can be subjected toone or more wash steps. If desired, the chromatographic material can becontacted with a solution of higher conductivity to desorbprocess-related impurities and product-related substances that havebound to the chromatographic material. In order to elute boundpolypeptides, the salt concentration of the buffer can be increased.

Ion exchange chromatography separates molecules based on differencesbetween the local charges of the proteins of interest and the localcharges of the chromatographic material. A packed ion-exchangechromatography column or an ion-exchange membrane device can be operatedeither in bind-elute mode or flow-through mode. In the bind-elute mode,the column or the membrane device is first conditioned with a bufferwith low ionic strength and proper pH under which the protein carriessufficient local opposite charge to the local charge of the materialimmobilized on the resin based matrix. During the feed load, the proteinof interest will be adsorbed to the resin due to electrostaticattraction. After washing the column or the membrane device with theequilibration buffer or another buffer with different pH and/orconductivity, the product recovery is achieved by increasing the ionicstrength (i.e., conductivity) of the elution buffer to compete with thesolute for the charged sites of the ion exchange matrix. Changing the pHand thereby altering the charge of the solute is another way to achieveelution of the solute. The change in conductivity or pH may be gradual(gradient elution) or stepwise (step elution). In the flow-through mode,the column or the membrane device is operated at selected pH andconductivity such that the protein of interest does not bind to theresin or the membrane while the process-related impurities and/orproduct-related substances will be retained to the column or themembrane. The column is then regenerated before next use.

Anionic or cationic substituents may be attached to matrices in order toform anionic or cationic supports for chromatography. Non-limitingexamples of anionic exchange substituents include diethylaminoethyl(DEAE), quaternary aminoethyl (QAE) and quaternary amine (O) groups.Cationic substitutents include carboxymethyl (CM), sulfoethyl (SE),sulfopropyl (SP), phosphate (P) and sulfonate (S). Cellulose ionexchange resins such as DE23™, DE32™, DE52™, CM-23™, CM-32™, and CM-52™are available from Whatman Ltd. Maidstone, Kent, U.K. SEPHADEX®-basedand -locross-linked ion exchangers are also known. For example, DEAE-,QAE-, CM-, and SP—SEPHADEX® and DEAE-, Q-, CM- and S-SEPHAROSE® andSEPHAROSE® Fast Flow, and Capto™ S are all available from GE Healthcare.Further, both DEAE and CM derivitized ethylene glycol-methacrylatecopolymer such as TOYOPEARL™ DEAE-6505 or M and TOYOPEARL™ CM-650S or Mare available from Toso Haas Co., Philadelphia, Pa., or Nuvia S andUNOSphere™ S from BioRad, Hercules, Calif., Eshmuno® S from EMDMillipore, Billerica, Calif.

This ion exchange step facilitates the purification of the antibody ofinterest by reducing impurities such as HCPs, DNA and aggregates. Incertain aspects, the ion exchange column is an anion exchange column.For example, but not by way of limitation, a suitable resin for such ananion exchange column is Capto™ Q, Nuvia™ Q, Q Sepharose Fast Flow, andPoros HQ 50. These resins are available from commercial sources such asGE Healthcare, BioRad, or Life Technologies. This anion exchangechromatography process can be carried out at or around room temperature.

4. Mixed Mode Chromatography

Mixed mode chromatography, also referred to herein as “multimodalchromatography”, is a chromatographic strategy that utilizes a supportcomprising a ligand that is capable of providing at least two different,in certain embodiments co-operative, sites that interact with thesubstance to be bound. In certain embodiments, one of these sites givesan attractive type of charge-charge interaction between the ligand andthe substance of interest and the other site provides for electronacceptor-donor interaction and/or hydrophobic and/or hydrophilicinteractions. Electron donor-acceptor interactions include interactionssuch as hydrogen-bonding, π-π, cation-π, charge transfer, dipole-dipole,induced dipole etc. Mixed mode chromatographic supports include, but arenot limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M, and Eshmuno® HCX.Mixed mode chromatography can be combined with one or more other stepsdescribed herein, including, but not limited to, Protein Achromatography, ion exchange, mixed mode chromatography, hydrophobicinteraction chromatography, etc.

In certain embodiments, the mixed mode chromatography resin is comprisedof ligands coupled to an organic or inorganic support, sometimes denoteda base matrix, directly or via a spacer. The support may be in the formof particles, such as essentially spherical particles, a monolith,filter, membrane, surface, capillaries, etc. In certain embodiments, thesupport is prepared from a native polymer, such as cross-linkedcarbohydrate material, such as agarose, agar, cellulose, dextran,chitosan, konjac, carrageenan, gellan, alginate etc. To obtain highadsorption capacities, the support can be porous, and ligands are thencoupled to the external surfaces as well as to the pore surfaces. Suchnative polymer supports can be prepared according to standard methods,such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta79(2), 393-398 (1964). Alternatively, the support can be prepared from asynthetic polymer, such as cross-linked synthetic polymers, e.g.,styrene or styrene derivatives, divinylbenzene, acrylamides, acrylateesters, methacrylate esters, vinyl esters, vinyl amides etc. Suchsynthetic polymers can be produced according to standard methods, seee.g., “Styrene based polymer supports developed by suspensionpolymerization” (R Arshady: Chimica e L′Industria 70(9), 70-75 (1988)).Porous native or synthetic polymer supports are also available fromcommercial sources, such as Amersham Biosciences, Uppsala, Sweden.

5. Hydrophobic Interaction Chromatography

The antibodies of the invention containing a PGPK modification, e.g.,anti-TNFα antibody, or antigen-binding portion thereof, may be purifiedby using a hydrophobic interaction chromatography (HIC) step in additionto a displacement chromatography step.

In performing the separation, the sample mixture is contacted with theHIC material, e.g., using a batch purification technique or using acolumn or membrane chromatography. Prior to HIC purification it may bedesirable to adjust the concentration of the kosmotropic salt to achievedesired protein binding to the resin or the membrane.

Whereas ion exchange chromatography relies on the local charge of theprotein of interest for selective separation, hydrophobic interactionchromatography employs the hydrophobic properties of the proteins toachieve selective separation. Hydrophobic groups on the protein interactwith hydrophobic groups of the resin or the membrane. The morehydrophobic a protein is the stronger it will interact with the columnor the membrane. Thus the HIC step removes process-related impurities(e.g., HCPs) as well as product-related substances (e.g., aggregates andfragments).

Like ion exchange chromatography, a HIC column or membrane device canalso be operated in product a bind-elute mode, a flow-through, or ahybrid mode wherein the product exhibits reversible binding to thechromatographic material. The bind-elute mode of operation has beenexplained above. For flow-through, the protein sample typically containsa relatively low level of salt than that used in the bind-elute mode.During this loading process, process-related impurities andproduct-related substances will bind to the resin while product flowsthrough the column. After loading, the column is regenerated with waterand cleaned with caustic solution to remove the bound impurities beforenext use. When used in connection with a hybrid mode, the product can beimmobilized on the chromatographic support in the presence of a loadingbuffer, but can be removed by successive washes of buffer identical toor substantially similar to the loading buffer. During this process,process-related impurities and product-relates substances will eitherbind to the chromatographic material or flow through with a profiledistinct from the protein of interest.

As hydrophobic interactions are strongest at high ionic strength, thisform of separation is conveniently performed following salt elutionstep, such as those that are typically used in connection with ionexchange chromatography. Alternatively, salts can be added into a lowsalt level feed stream before this step. Adsorption of the antibody to aHIC column is favored by high salt concentrations, but the actualconcentrations can vary over a wide range depending on the nature of theprotein of interest, salt type and the particular HIC ligand chosen.Various ions can be arranged in a so-called soluphobic series dependingon whether they promote hydrophobic interactions (salting-out effects)or disrupt the structure of water (chaotropic effect) and lead to theweakening of the hydrophobic interaction. Cations are ranked in terms ofincreasing salting out effect as Ba²⁺; Ca²⁺; Mg²⁺; Li⁺; Cs⁺; Na⁺; K⁺;Rb⁺; NH₄ ⁺, while anions may be ranked in terms of increasing chaotropiceffect as PO₄ ³⁻; SO₄ ²⁻; CH₃CO₃ ⁻; Cl⁻; Br⁻; NO₃ ⁻; ClO₄ ⁻; I⁻; SCN⁻.

In general, Na⁺, K⁺ or NH₄ ⁺ sulfates effectively promote ligand-proteininteraction in HIC. Salts may be formulated that influence the strengthof the interaction as given by the following relationship:(NH₄)₂SO₄>Na₂SO₄>NaCl>NH₄Cl>NaBr>NaSCN. In general, salt concentrationsof between about 0.75 M and about 2 M ammonium sulfate or between about1 and 4 M NaCl are useful.

HIC media normally comprise a base matrix (e.g., cross-linked agarose orsynthetic copolymer material) to which hydrophobic ligands (e.g., alkylor aryl groups) are coupled. A suitable HIC media comprises an agaroseresin or a membrane functionalized with phenyl groups (e.g., a PhenylSepharose™ from GE Healthcare or a Phenyl Membrane from Sartorius). ManyHIC resins are available commercially. Examples include, but are notlimited to, Capto Phenyl, Phenyl Sepharose™ 6 Fast Flow with low or highsubstitution, Phenyl Sepharose™ High Performance, Octyl Sepharose™ HighPerformance (GE Healthcare); Fractogel™ EMD Propyl or Fractogel™ EMDPhenyl (E. Merck, Germany); Macro-Prep™ Methyl or Macro-Prep™ t-Butylcolumns (Bio-Rad, California); WP HI-Propyl (C3)™ (J. T. Baker, NewJersey); and Toyopearl™ ether, phenyl or butyl (TosoHaas, Pa.).

6. Viral Filtration

Viral filtration is a dedicated viral reduction step in the entirepurification process. This step is usually performed postchromatographic polishing steps. Viral reduction can be achieved via theuse of suitable filters including, but not limited to, Planova 20N™, 50N or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMDMillipore, ViroSart CPV from Sartorius, or Ultipor DV20 or DV50™ filterfrom Pall Corporation. It will be apparent to one of ordinary skill inthe art to select a suitable filter to obtain desired filtrationperformance.

7. Ultrafiltration/Diafiltration

Certain embodiments of the present invention employ ultrafiltration anddiafiltration steps to further concentrate and formulate the protein ofinterest, e.g., an antibody of the invention. Ultrafiltration isdescribed in detail in: Microfiltration and Ultrafiltration: Principlesand Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York,N.Y., 1996); and in: Ultrafiltration Handbook, Munir Cheryan (TechnomicPublishing, 1986; ISBN No. 87762-456-9). One filtration process isTangential Flow Filtration as described in the Millipore catalogueentitled “Pharmaceutical Process Filtration Catalogue” pp. 177-202(Bedford, Mass., 1995/96). In contrast, diafiltration is a method ofusing membrane filters to remove and exchange salts, sugars, andnon-aqueous solvents, to separate free from bound species, to remove lowmolecular-weight species, and/or to cause the rapid change of ionicand/or pH environments. Examples of membrane cassettes suitable for thepresent invention include, but not limited to, Pellicon 2 or Pellicon 3cassetts with 10 kD, 30 kD or 50 kD membranes from EMD Millipore, Kvick10 kD, 30 kD or 50 kD membrane cassettes from GE Healthcare, andCentramate or Centrasette 10 kD, 30 kD or 50 kD cassettes from PallCorporation.

8. Methods of Assaying Sample Purity

a. Assaying Host Cell Protein

The antibodies of the present invention containing a PGPK modificationmay be assayed for purity using methods known in the art and describedherein. For example, residual levels of host cell protein (HCP)concentration in the isolated/purified antibody composition may bemeasured. As described above, HCPs are desirably excluded from the finaltarget substance product. Exemplary HCPs include proteins originatingfrom the source of the antibody production. Failure to identify andsufficiently remove HCPs from the target antibody may lead to reducedefficacy and/or adverse subject reactions.

As used herein, the term “HCP ELISA” refers to an ELISA where the secondantibody used in the assay is specific to the HCPs produced from cells,e.g., CHO cells, used to generate the antibody of interest. The secondantibody may be produced according to conventional methods known tothose of skill in the art. For example, the second antibody may beproduced using HCPs obtained by sham production and purification runs,i.e., the same cell line used to produce the antibody of interest isused, but the cell line is not transfected with antibody DNA. In anexemplary embodiment, the second antibody is produced using HCPs similarto those expressed in the cell expression system of choice, i.e., thecell expression system used to produce the target antibody.

Generally, HCP ELISA comprises sandwiching a liquid sample comprisingHCPs between two layers of antibodies, i.e., a first antibody and asecond antibody. The sample is incubated during which time the HCPs inthe sample are captured by the first antibody, for example, but notlimited to goat anti-CHO, affinity purified (Cygnus). A labeled secondantibody, or blend of antibodies, specific to the HCPs produced from thecells used to generate the antibody, e.g., anti-CHO HCP Biotinylated, isadded, and binds to the HCPs within the sample. In certain embodimentsthe first and second antibodies are polyclonal antibodies. In certainaspects the first and second antibodies are blends of polyclonalantibodies raised against HCPs. The amount of HCP contained in thesample is determined using the appropriate test based on the label ofthe second antibody.

HCP ELISA may be used for determining the level of HCPs in an antibodycomposition, such as an eluate, displacement samples or flow-throughfractions obtained using the process described above. The presentinvention also provides a composition comprising an antibody, whereinthe composition has less than 100 ng/mgHCPs as determined by an HCPEnzyme Linked Immunosorbent Assay (“ELISA”).

b. Assaying Charge and Size Variants

In certain embodiments, the levels of product-related substances, suchas basic species, acidic species and other variants, in thechromatographic samples produced using the techniques described hereinare analyzed. In certain embodiments a CEX-HPLC method is employed. Forexample, but not by way of limitation, a 4 mm×250 mm analytical DionexProPac WCX-10 column (Dionex, Calif.) can be used along with a ShimazhuHPLC system. In certain embodiments, the mobile phases employed in suchan assay will include a 10 mM Sodium Phosphate dibasic pH 7.5 buffer(Mobile phase A) and a 10 mM Sodium Phosphate dibasic, 500 mM SodiumChloride pH 5.5 buffer (Mobile phase B). In certain embodiments, themobile phases can include a 20 mM MES, pH 6.5 buffer (Mobile phase A)and a 20 mM MES, 500 mM NaCl, pH 6.5 buffer (Mobile phase B). In certainembodiments, the mobile phases can include a 20 mM MES, pH 6.2 buffer(Mobile phase A) and a 20 mM MES, 250 mM NaCl, pH 6.2 buffer (Mobilephase B). In certain embodiments, a binary gradient, for example, butnot by way of limitation, a 6% B: 0 min; 6-16% B: 0-20 min; 16-100% B:20-22 min; 100% B: 22-26 min; 100-6% B: 26-28 min; 6% B: 28-35 mingradient can be used with detection at 280 nm. In certain, non-limitingembodiments, a binary gradient comprising 10% B: 0 min; 10-28% B: 1-46min; 28-100% B: 46-47 min; 100% B: 47-52 min; 100-10% B: 52-53 min; 10%B: 53-58 min, will be used with detection at 280 nm. In certainembodiments, a binary gradient such as a 1% B: 0-1 min; 1-25% B: 1-46min; 25-100% B: 46-47 min; 100% B: 47-52 min; 100-1% B: 52-53 min; 1% B:53-60 min gradient can be used with detection at 280 nm. Quantitationcan be based on the relative area percentage of detected peaks. Incertain embodiments, the peaks that elute at residence time less than ˜7min will represent the acidic peaks or AR region. In certainembodiments, all peaks eluting prior to the Main Isoform peak can besummed as the acidic region, and all peaks eluting after the Main peakcan be summed as the basic region. In certain embodiments, all peakseluting prior to the Main Isoform peak (but after, e.g., a 2 minretention time) were summed as the acidic region, and all peaks elutingafter the Main peak were summed as the basic region.

In certain embodiments, the levels of aggregates, monomer, and fragmentsin the chromatographic samples produced using the techniques describedherein are analyzed. In certain embodiments, the aggregates, monomer,and fragments are measured using a size exclusion chromatographic (SEC)method for each molecule. For example, but not by way of limitation, aTSK-gel G3000SWxL, 5 μm, 125 Å, 7.8×300 mm column (Tosoh Bioscience) canbe used in connection with certain embodiments, while a TSK-gel SuperSW3000, 4 μm, 250 Å, 4.6×300 mm column (Tosoh Bioscience) can be used inalternative embodiments. In certain embodiments, the aforementionedcolumns are used along with an Agilent or a Shimazhu HPLC system. Incertain embodiments, sample injections are made under isocratic elutionconditions using a mobile phase consisting of, for example, 100 mMsodium sulfate and 100 mM sodium phosphate at pH 6.8, and detected withUV absorbance at 214 nm. In certain embodiments, the mobile phase willconsist of 1×PBS at pH 7.4, and elution profile detected with UVabsorbance at 280 nm. In certain embodiments, quantification is based onthe relative area of detected peaks.

IV. Methods of Treatment

The antibodies, or antigen-binding portions thereof, of the inventioncomprising a PGPK modification are useful in treating diseases ordisorders. In one embodiment, the antibody, or antigen-binding portionthereof, is an anti-TNFα antibody, or antigen-binding portion thereof.TNFα has been implicated in the pathophysiology of a wide variety ofdisorders, including sepsis, infections, autoimmune diseases, transplantrejection and graft-versus-host disease (see e.g., Moeller, A., et al.(1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.;European Patent Publication No. 260 610 B1 by Moeller, A., et al.Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey, K. J. andCerami, A. (1994) Annu. Rev. Med. 45:491-503). The present inventionprovides methods of treating a subject having a disorder in which TNFαactivity is detrimental by administering a therapeutically effectiveamount of an antibody, or antigen-binding portion thereof, of theinvention comprising a PGPK modification to the subject, therebytreating the TNFα-associated disease or disorder. In one aspect, theTNFα is human TNFα and the subject is a human subject.

As used herein, the term “a disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of TNFα in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to anexcerbation of the disorder. Accordingly, a disorder in which TNFαactivity is detrimental is a disorder in which inhibition of TNFαactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of TNFα in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofTNFα in serum, plasma, synovial fluid, etc. of the subject), which canbe detected, for example, using an anti-TNFα antibody. Disorders inwhich TNFα activity is detrimental are well known in the art anddescribed in detail in U.S. Pat. No. 8,231,876, the entire contents ofwhich are expressly incorporated herein by reference. Disorders in whichTNFα activity is detrimental are also described in “Highlights ofPrescribing Information” for HUMIRA® (adalimumab) Injection (RevisedJanuary 2008).

In one embodiment, “a disorder in which TNFα activity is detrimental”includes sepsis (including septic shock, endotoxic shock, gram negativesepsis and toxic shock syndrome), autoimmune diseases (includingrheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and goutyarthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmuneuveitis, nephrotic syndrome, multisystem autoimmune diseases, lupus(including systemic lupus, lupus nephritis and lupus cerebritis),Crohn's disease and autoimmune hearing loss), infectious diseases(including malaria, meningitis, acquired immune deficiency syndrome(AIDS), influenza and cachexia secondary to infection), allograftrejection and graft versus host disease, malignancy, pulmonary disorders(including adult respiratory distress syndrome (ARDS), shock lung,chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonaryfibrosis, silicosis, idiopathic interstitial lung disease and chronicobstructive airway disorders (COPD), such as asthma), intestinaldisorders (including inflammatory bowel disorders, idiopathicinflammatory bowel disease, Crohn's disease and Crohn's disease-relateddisorders (including fistulas in the bladder, vagina, and skin; bowelobstructions; abscesses; nutritional deficiencies; complications fromcorticosteroid use; inflammation of the joints; erythem nodosum;pyoderma gangrenosum; lesions of the eye, Crohn's related arthralgias,fistulizing Crohn's indeterminant colitis and pouchitis), cardiacdisorders (including ischemia of the heart, heart insufficiency,restenosis, congestive heart failure, coronary artery disease, anginapectoris, myocardial infarction, cardiovascular tissue damage caused bycardiac arrest, cardiovascular tissue damage caused by cardiac bypass,cardiogenic shock, and hypertension, atherosclerosis, cardiomyopathy,coronary artery spasm, coronary artery disease, valvular disease,arrhythmias, and cardiomyopathies), spondyloarthropathies (includingankylosing spondylitis, psoriatic arthritis/spondylitis, enteropathicarthritis, reactive arthritis or Reiter's syndrome, and undifferentiatedspondyloarthropathies), metabolic disorders (including obesity anddiabetes, including type 1 diabetes mellitus, type 2 diabetes mellitus,diabetic neuropathy, peripheral neuropathy, diabetic retinopathy,diabetic ulcerations, retinopathy ulcerations and diabeticmacrovasculopathy), anemia, pain (including acute and chronic pains,such as neuropathic pain and post-operative pain, chronic lower backpain, cluster headaches, herpes neuralgia, phantom limb pain, centralpain, dental pain, opioid-resistant pain, visceral pain, surgical pain,bone injury pain, pain during labor and delivery, pain resulting fromburns, including sunburn, post partum pain, migraine, angina pain, andgenitourinary tract-related pain including cystitis), hepatic disorders(including hepatitis, alcoholic hepatitis, viral hepatitis, alcoholiccirrhosis, al antitypsin deficiency, autoimmune cirrhosis, cryptogeniccirrhosis, fulminant hepatitis, hepatitis B and C, and steatohepatitis,cystic fibrosis, primary biliary cirrhosis, sclerosing cholangitis andbiliary obstruction), skin and nail disorders (including psoriasis(including chronic plaque psoriasis, guttate psoriasis, inversepsoriasis, pustular psoriasis and other psoriasis disorders), pemphigusvulgaris, scleroderma, atopic dermatitis (eczema), sarcoidosis, erythemanodosum, hidradenitis suppurative, lichen planus, Sweet's syndrome,scleroderma and vitiligo), vasculitides (including Behcet's disease),and other disorders, such as juvenile rheumatoid arthritis (JRA),endometriosis, prostatitis, choroidal neovascularization, sciatica,Sjogren's syndrome, uveitis, wet macular degeneration, osteoporosis,osteoarthritis, active axial spondyloarthritis (active axSpA) andnon-radiographic axial spondyloarthritis (nr-axSpA).

As used herein, the term “subject” is intended to include livingorganisms, e.g., prokaryotes and eukaryotes. Examples of subjectsinclude mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats, and transgenic non-human animals. In specificembodiments of the invention, the subject is a human.

As used herein, the term “treatment” or “treat” refers to boththerapeutic treatment and prophylactic or preventative measures. Thosein need of treatment include those already with the disorder, as well asthose in which the disorder is to be prevented.

The term “dosing” or “dose” or “dosage”, as used herein, refers to theadministration of a substance (e.g., an antibody of interest, forexample, an anti-TNFα antibody, or antigen-binding portion thereof) toachieve a therapeutic objective (e.g., the treatment or amelioration ofa symptom of a disease or disorder).

In one embodiment, the invention provides a method of administering acomposition comprising an anti-TNFα antibody, or antigen binding portionthereof, comprising a PGPK modification to a subject such that TNFαactivity is inhibited or a disorder in which TNFα activity isdetrimental is treated. In one aspect, the TNFα is human TNFα and thesubject is a human subject. In one embodiment, the anti-TNFα antibody isadalimumab, also referred to as HUMIRA®.

The compositions comprising an antibody, or antigen-binding portionthereof, comprising a PGPK modification can be administered by a varietyof methods known in the art. Exemplary routes/modes of administrationinclude subcutaneous injection, intravenous injection or infusion. Incertain aspects, a composition comprising an antibody, orantigen-binding portion thereof, containing a PGPK modification may beorally administered. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. In certainembodiments, it is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit comprising a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic or prophylactic effect to be achieved, and(b) the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a composition comprising anantibody, or antigen-binding portion thereof, comprising a PGPKmodification of the invention is 0.01-20 mg/kg, or 1-10 mg/kg, or 0.3-1mg/kg. With respect to compositions of the invention comprising ananti-TNFα antibody, or antigen-binding portion thereof, such asadalimumab, comprising a PGPK modification, an exemplary dose is 40 mgevery other week. In some embodiments, in particular for treatment ofulcerative colitis or Crohn's disease, an exemplary dose includes aninitial dose (Day 1) of 160 mg (e.g., four 40 mg injections in one dayor two 40 mg injections per day for two consecutive days), a second dosetwo weeks later of 80 mg, and a maintenance dose of 40 mg every otherweek beginning two weeks later. Alternatively, for psoriasis, forexample, a dosage can include an 80 mg initial dose followed by 40 mgevery other week starting one week after the initial dose.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

V. Pharmaceutical Formulations of Antibodies of the Invention

The present invention further provides preparations and formulationscomprising the antibodies (including antibody fragments) comprising aPGPK modification of the present invention. It should be understood thatany of the antibodies and antibody fragments described herein, includingantibodies and antibody fragments having any one or more of thestructural and functional features described in detail throughout theapplication, may be formulated or prepared as described below. Whenvarious formulations are described in this section as including anantibody, it is understood that such an antibody may be an antibody oran antibody fragment having any one or more of the characteristics ofthe antibodies and antibody fragments described herein. In oneembodiment, the antibody is an anti-TNFα antibody, or antigen-bindingportion thereof.

In one embodiment, a composition of the invention comprising anantibody, or antigen-binding portion thereof, comprising a PGPKmodification is formulated with the same or similar excipients andbuffers as are present in the commercial adalimumab (HUMIRA®)formulation, as described in the HUMIRA® Prescribing Information, whichis expressly incorporated herein by reference. For example, eachprefilled syringe of HUMIRA®, which is administered subcutaneously,delivers 0.8 mL (40 mg) of drug product to the subject. Each 0.8 mL ofHUMIRA® contains 40 mg adalimumab, 4.93 mg sodium chloride, 0.69 mgmonobasic sodium phosphate dehydrate, 1.22 mg dibasic sodium phosphatedehydrate, 0.24 mg sodium citrate, 1.04 mg citric acid monohydrate, 9.6mg mannitol, 0.8 mg polysorbate 80, and water for Injection, USP. Sodiumhydroxide is added as necessary to adjust pH.

In certain embodiments, the antibodies of the invention may beformulated with a pharmaceutically acceptable carrier as pharmaceutical(therapeutic) compositions, and may be administered by a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. The term “pharmaceutically acceptable carrier” meansone or more non-toxic materials that do not interfere with theeffectiveness of the biological activity of the active ingredients. Suchpreparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. Such pharmaceutically acceptable preparations may also routinelycontain compatible solid or liquid fillers, diluents or encapsulatingsubstances which are suitable for administration into a human. The term“carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being co-mingled with the antibodies of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficacy.

The formulations of the invention are present in a form known in the artand acceptable for therapeutic uses. In one embodiment, a formulation ofthe invention is a liquid formulation. In another embodiment, aformulation of the invention is a lyophilized formulation. In a furtherembodiment, a formulation of the invention is a reconstituted liquidformulation. In one embodiment, a formulation of the invention is astable liquid formulation. In one embodiment, a liquid formulation ofthe invention is an aqueous formulation. In another embodiment, theliquid formulation is non-aqueous. In a specific embodiment, a liquidformulation of the invention is an aqueous formulation wherein theaqueous carrier is distilled water.

The formulations of the invention comprise an antibody in aconcentration resulting in a w/v appropriate for a desired dose. Theantibody may be present in the formulation at a concentration of about 1mg/ml to about 500 mg/ml, e.g., at a concentration of at least 1 mg/ml,at least 5 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 20mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 35 mg/ml, at least40 mg/ml, at least 45 mg/ml, at least 50 mg/ml, at least 55 mg/ml, atleast 60 mg/ml, at least 65 mg/ml, at least 70 mg/ml, at least 75 mg/ml,at least 80 mg/ml, at least 85 mg/ml, at least 90 mg/ml, at least 95mg/ml, at least 100 mg/ml, at least 105 mg/ml, at least 110 mg/ml, atleast 115 mg/ml, at least 120 mg/ml, at least 125 mg/ml, at least 130mg/ml, at least 135 mg/ml, at least 140 mg/ml, at least 150 mg/ml, atleast 200 mg/ml, at least 250 mg/ml, or at least 300 mg/ml.

In a specific embodiment, a formulation of the invention comprises atleast about 100 mg/ml, at least about 125 mg/ml, at least 130 mg/ml, orat least about 150 mg/ml of an antibody of the invention.

In one embodiment, the concentration of antibody, which is included inthe formulation of the invention, is between about 1 mg/ml and about 25mg/ml, between about 1 mg/ml and about 200 mg/ml, between about 25 mg/mland about 200 mg/ml, between about 50 mg/ml and about 200 mg/ml, betweenabout 75 mg/ml and about 200 mg/ml, between about 100 mg/ml and about200 mg/ml, between about 125 mg/ml and about 200 mg/ml, between about150 mg/ml and about 200 mg/ml, between about 25 mg/ml and about 150mg/ml, between about 50 mg/ml and about 150 mg/ml, between about 75mg/ml and about 150 mg/ml, between about 100 mg/ml and about 150 mg/ml,between about 125 mg/ml and about 150 mg/ml, between about 25 mg/ml andabout 125 mg/ml, between about 50 mg/ml and about 125 mg/ml, betweenabout 75 mg/ml and about 125 mg/ml, between about 100 mg/ml and about125 mg/ml, between about 25 mg/ml and about 100 mg/ml, between about 50mg/ml and about 100 mg/ml, between about 75 mg/ml and about 100 mg/ml,between about 25 mg/ml and about 75 mg/ml, between about 50 mg/ml andabout 75 mg/ml, or between about 25 mg/ml and about 50 mg/ml.

In a specific embodiment, a formulation of the inventions comprisesbetween about 90 mg/ml and about 110 mg/ml or between about 100 mg/mland about 210 mg/ml of an antibody.

The formulations of the invention comprising an antibody may furthercomprise one or more active compounds as necessary for the particularindication being treated, typically those with complementary activitiesthat do not adversely affect each other. Such additional activecompound/s is/are suitably present in combination in amounts that areeffective for the purpose intended.

The formulations of the invention may be prepared for storage by mixingthe antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers,including, but not limited to buffering agents, saccharides, salts,surfactants, solubilizers, polyols, diluents, binders, stabilizers,salts, lipophilic solvents, amino acids, chelators, preservatives, orthe like (Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 12^(th) edition, L. Brunton, et al. Remington'sPharmaceutical Sciences, 16th edition, Osol, A. Ed. (1999)), in the formof lyophilized formulations or aqueous solutions at a desired finalconcentration. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as histidine, phosphate, citrate, glycine, acetateand other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrolidone; aminoacids such as glycine, glutamine, asparagine, histidine, arginine, orlysine; monosaccharides, disaccharides, and other carbohydratesincluding trehalose, glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;salt-forming counter-ions such as sodium; metal complexes (e.g.,Zn-protein complexes); and/or non-ionic surfactants such as TWEEN,polysorbate 80, PLURONICS™ or polyethylene glycol (PEG).

The buffering agent may be histidine, citrate, phosphate, glycine, oracetate. The saccharide excipient may be trehalose, sucrose, mannitol,maltose or raffinose. The surfactant may be polysorbate 20, polysorbate40, polysorbate 80, or Pluronic F68. The salt may be NaCl, KCl, MgCl₂,or CaCl₂.

The formulations of the invention may include a buffering or pHadjusting agent to provide improved pH control. A formulation of theinvention may have a pH of between about 3.0 and about 9.0, betweenabout 4.0 and about 8.0, between about 5.0 and about 8.0, between about5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.5and about 8.0, between about 5.5 and about 7.0, or between about 5.5 andabout 6.5. In a further embodiment, a formulation of the invention has apH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1,about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4,about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about7.5, about 8.0, about 8.5, or about 9.0. In a specific embodiment, aformulation of the invention has a pH of about 6.0. One of skill in theart understands that the pH of a formulation generally should not beequal to the isoelectric point of the particular antibody to be used inthe formulation.

Typically, the buffering agent is a salt prepared from an organic orinorganic acid or base. Representative buffering agents include, but arenot limited to, organic acid salts such as salts of citric acid,ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinicacid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride,or phosphate buffers. In addition, amino acid components can alsofunction in a buffering capacity. Representative amino acid componentswhich may be utilized in the formulations of the invention as bufferingagents include, but are not limited to, glycine and histidine. Incertain embodiments, the buffering agent is chosen from histidine,citrate, phosphate, glycine, and acetate. In a specific embodiment, thebuffering agent is histidine. In another specific embodiment, thebuffering agent is citrate. In yet another specific embodiment, thebuffering agent is glycine. The purity of the buffering agent should beat least 98%, or at least 99%, or at least 99.5%. As used herein, theterm “purity” in the context of histidine and glycine refers to chemicalpurity of histidine or glycine as understood in the art, e.g., asdescribed in The Merck Index, 13^(th) ed., O'Neil et al. ed. (Merck &Co., 2001).

Buffering agents are typically used at concentrations between about 1 mMand about 200 mM or any range or value therein, depending on the desiredionic strength and the buffering capacity required. The usualconcentrations of conventional buffering agents employed in parenteralformulations can be found in: Pharmaceutical Dosage Form: ParenteralMedications, Volume 1, 2^(nd) Edition, Chapter 5, p. 194, De Luca andBoylan, “Formulation of Small Volume Parenterals”, Table 5: Commonlyused additives in Parenteral Products. In one embodiment, the bufferingagent is at a concentration of about 1 mM, or of about 5 mM, or of about10 mM, or of about 15 mM, or of about 20 mM, or of about 25 mM, or ofabout 30 mM, or of about 35 mM, or of about 40 mM, or of about 45 mM, orof about 50 mM, or of about 60 mM, or of about 70 mM, or of about 80 mM,or of about 90 mM, or of about 100 mM. In one embodiment, the bufferingagent is at a concentration of 1 mM, or of mM, or of 10 mM, or of 15 mM,or of 20 mM, or of 25 mM, or of 30 mM, or of 35 mM, or of 40 mM, or of45 mM, or of 50 mM, or of 60 mM, or of 70 mM, or of 80 mM, or of 90 mM,or of 100 mM. In a specific embodiment, the buffering agent is at aconcentration of between about 5 mM and about 50 mM. In another specificembodiment, the buffering agent is at a concentration of between 5 mMand 20 mM.

In certain embodiments, the formulation of the invention compriseshistidine as a buffering agent. In one embodiment the histidine ispresent in the formulation of the invention at a concentration of atleast about 1 mM, at least about 5 mM, at least about 10 mM, at leastabout 20 mM, at least about 30 mM, at least about 40 mM, at least about50 mM, at least about 75 mM, at least about 100 mM, at least about 150mM, or at least about 200 mM histidine. In another embodiment, aformulation of the invention comprises between about 1 mM and about 200mM, between about 1 mM and about 150 mM, between about 1 mM and about100 mM, between about 1 mM and about 75 mM, between about 10 mM andabout 200 mM, between about 10 mM and about 150 mM, between about 10 mMand about 100 mM, between about 10 mM and about 75 mM, between about 10mM and about 50 mM, between about 10 mM and about 40 mM, between about10 mM and about 30 mM, between about 20 mM and about 75 mM, betweenabout 20 mM and about 50 mM, between about 20 mM and about 40 mM, orbetween about 20 mM and about 30 mM histidine. In a further embodiment,the formulation comprises about 1 mM, about 5 mM, about 10 mM, about 20mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM,about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about100 mM, about 150 mM, or about 200 mM histidine. In a specificembodiment, a formulation may comprise about 10 mM, about 25 mM, or nohistidine.

The formulations of the invention may comprise a carbohydrate excipient.Carbohydrate excipients can act, e.g., as viscosity enhancing agents,stabilizers, bulking agents, solubilizing agents, and/or the like.Carbohydrate excipients are generally present at between about 1% toabout 99% by weight or volume, e.g., between about 0.1% to about 20%,between about 0.1% to about 15%, between about 0.1% to about 5%, betweenabout 1% to about 20%, between about 5% to about 15%, between about 8%to about 10%, between about 10% and about 15%, between about 15% andabout 20%, between 0.1% to 20%, between 5% to 15%, between 8% to 10%,between 10% and 15%, between 15% and 20%, between about 0.1% to about5%, between about 5% to about 10%, or between about 15% to about 20%. Instill other specific embodiments, the carbohydrate excipient is presentat 1%, or at 1.5%, or at 2%, or at 2.5%, or at 3%, or at 4%, or at 5%,or at 10%, or at 15%, or at 20%.

Carbohydrate excipients suitable for use in the formulations of theinvention include, but are not limited to, monosaccharides such asfructose, maltose, galactose, glucose, D-mannose, sorbose, and the like;disaccharides, such as lactose, sucrose, trehalose, cellobiose, and thelike; polysaccharides, such as raffinose, melezitose, maltodextrins,dextrans, starches, and the like; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like.In one embodiment, the carbohydrate excipients for use in the presentinvention are chosen from, sucrose, trehalose, lactose, mannitol, andraffinose. In a specific embodiment, the carbohydrate excipient istrehalose. In another specific embodiment, the carbohydrate excipient ismannitol. In yet another specific embodiment, the carbohydrate excipientis sucrose. In still another specific embodiment, the carbohydrateexcipient is raffinose. The purity of the carbohydrate excipient shouldbe at least 98%, or at least 99%, or at least 99.5%.

In a specific embodiment, the formulations of the invention may comprisetrehalose. In one embodiment, a formulation of the invention comprisesat least about 1%, at least about 2%, at least about 4%, at least about8%, at least about 20%, at least about 30%, or at least about 40%trehalose. In another embodiment, a formulation of the inventioncomprises between about 1% and about 40%, between about 1% and about30%, between about 1% and about 20%, between about 2% and about 40%,between about 2% and about 30%, between about 2% and about 20%, betweenabout 4% and about 40%, between about 4% and about 30%, or between about4% and about 20% trehalose. In a further embodiment, a formulation ofthe invention comprises about 1%, about 2%, about 4%, about 6%, about8%, about 15%, about 20%, about 30%, or about 40% trehalose. In aspecific embodiment, a formulation of the invention comprises about 4%,about 6% or about 15% trehalose.

In certain embodiments, a formulation of the invention comprises anexcipient. In a specific embodiment, a formulation of the inventioncomprises at least one excipient chosen from: sugar, salt, surfactant,amino acid, polyol, chelating agent, emulsifier and preservative. In oneembodiment, a formulation of the invention comprises a salt, e.g., asalt selected from: NaCl, KCl, CaCl₂, and MgCl₂. In a specificembodiment, the formulation comprises NaCl.

A formulation of the invention may comprise at least about 10 mM, atleast about 25 mM, at least about 50 mM, at least about 75 mM, at leastabout 80 mM, at least about 100 mM, at least about 125 mM, at leastabout 150 mM, at least about 175 mM, at least about 200 mM, or at leastabout 300 mM sodium chloride (NaCl). In a further embodiment, theformulation may comprise between about 10 mM and about 300 mM, betweenabout 10 mM and about 200 mM, between about 10 mM and about 175 mM,between about 10 mM and about 150 mM, between about 25 mM and about 300mM, between about 25 mM and about 200 mM, between about 25 mM and about175 mM, between about 25 mM and about 150 mM, between about 50 mM andabout 300 mM, between about 50 mM and about 200 mM, between about 50 mMand about 175 mM, between about 50 mM and about 150 mM, between about 75mM and about 300 mM, between about 75 mM and about 200 mM, between about75 mM and about 175 mM, between about 75 mM and about 150 mM, betweenabout 100 mM and about 300 mM, between about 100 mM and about 200 mM,between about 100 mM and about 175 mM, or between about 100 mM and about150 mM sodium chloride. In a further embodiment, the formulation maycomprise about 10 mM, about 25 mM, about 50 mM, about 75 mM, about 80mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200mM, or about 300 mM sodium chloride.

A formulation of the invention may also comprise an amino acid, e.g.,lysine, arginine, glycine, histidine or an amino acid salt. Theformulation may comprise at least about 1 mM, at least about 10 mM, atleast about 25 mM, at least about 50 mM, at least about 100 mM, at leastabout 150 mM, at least about 200 mM, at least about 250 mM, at leastabout 300 mM, at least about 350 mM, or at least about 400 mM of anamino acid. In another embodiment, the formulation may comprise betweenabout 1 mM and about 100 mM, between about 10 mM and about 150 mM,between about 25 mM and about 250 mM, between about 25 mM and about 300mM, between about 25 mM and about 350 mM, between about 25 mM and about400 mM, between about 50 mM and about 250 mM, between about 50 mM andabout 300 mM, between about 50 mM and about 350 mM, between about 50 mMand about 400 mM, between about 100 mM and about 250 mM, between about100 mM and about 300 mM, between about 100 mM and about 400 mM, betweenabout 150 mM and about 250 mM, between about 150 mM and about 300 mM, orbetween about 150 mM and about 400 mM of an amino acid. In a furtherembodiment, a formulation of the invention comprises about 1 mM, 1.6 mM,25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250mM, about 300 mM, about 350 mM, or about 400 mM of an amino acid.

The formulations of the invention may further comprise a surfactant. Theterm “surfactant” as used herein refers to organic substances havingamphipathic structures; namely, they are composed of groups of opposingsolubility tendencies, typically an oil-soluble hydrocarbon chain and awater-soluble ionic group. Surfactants can be classified, depending onthe charge of the surface-active moiety, into anionic, cationic, andnonionic surfactants. Surfactants are often used as wetting,emulsifying, solubilizing, and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.Pharmaceutically acceptable surfactants like polysorbates (e.g.,polysorbates 20 or 80); polyoxamers (e.g., poloxamer 188); Triton;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUA™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., PLURONICS™, PF68, etc.), canoptionally be added to the formulations of the invention to reduceaggregation. In one embodiment, a formulation of the invention comprisesPolysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate 80.Surfactants are particularly useful if a pump or plastic container isused to administer the formulation. The presence of a pharmaceuticallyacceptable surfactant mitigates the propensity for the protein toaggregate. The formulations may comprise a polysorbate which is at aconcentration ranging from between about 0.001% to about 1%, or about0.001% to about 0.1%, or about 0.01% to about 0.1%. In other specificembodiments, the formulations of the invention comprise a polysorbatewhich is at a concentration of 0.001%, or 0.002%, or 0.003%, or 0.004%,or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or0.015%, or 0.02%.

The formulations of the invention may optionally further comprise othercommon excipients and/or additives including, but not limited to,diluents, binders, stabilizers, lipophilic solvents, preservatives,adjuvants, or the like. Pharmaceutically acceptable excipients and/oradditives may be used in the formulations of the invention. Commonlyused excipients/additives, such as pharmaceutically acceptable chelators(for example, but not limited to, EDTA, DTPA or EGTA) can optionally beadded to the formulations of the invention to reduce aggregation. Theseadditives are particularly useful if a pump or plastic container is usedto administer the formulation.

Preservatives, such as phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (for example, but notlimited to, hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl andthe like), benzalkonium chloride, benzethonium chloride, sodiumdehydroacetate and thimerosal, or mixtures thereof can optionally beadded to the formulations of the invention at any suitable concentrationsuch as between about 0.001% to about 5%, or any range or value therein.The concentration of preservative used in the formulations of theinvention is a concentration sufficient to yield a microbial effect.Such concentrations are dependent on the preservative selected and arereadily determined by the skilled artisan.

Other contemplated excipients/additives, which may be utilized in theformulations of the invention include, for example, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,lipids such as phospholipids or fatty acids, steroids such ascholesterol, protein excipients such as serum albumin (human serumalbumin (HSA), recombinant human albumin (rHA)), gelatin, casein,salt-forming counterions such as sodium and the like. These andadditional known pharmaceutical excipients and/or additives suitable foruse in the formulations of the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 21^(st) ed.,Lippincott Williams & Wilkins, (2005), and in the “Physician's DeskReference”, 60^(th) ed., Medical Economics, Montvale, N.J. (2005).Pharmaceutically acceptable carriers can be routinely selected that aresuitable for the mode of administration, solubility and/or stability ofan antibody, as well known those in the art or as described herein.

It will be understood by one skilled in the art that the formulations ofthe invention may be isotonic with human blood, wherein the formulationsof the invention have essentially the same osmotic pressure as humanblood. Such isotonic formulations will generally have an osmoticpressure from about 250 mOSm to about 350 mOSm. Isotonicity can bemeasured by, for example, using a vapor pressure or ice-freezing typeosmometer. Tonicity of a formulation is adjusted by the use of tonicitymodifiers. “Tonicity modifiers” are those pharmaceutically acceptableinert substances that can be added to the formulation to provide anisotonity of the formulation. Tonicity modifiers suitable for thisinvention include, but are not limited to, saccharides, salts and aminoacids.

In certain embodiments, the formulations of the present invention havean osmotic pressure from about 100 mOSm to about 1200 mOSm, or fromabout 200 mOSm to about 1000 mOSm, or from about 200 mOSm to about 800mOSm, or from about 200 mOSm to about 600 mOSm, or from about 250 mOSmto about 500 mOSm, or from about 250 mOSm to about 400 mOSm, or fromabout 250 mOSm to about 350 mOSm.

The concentration of any one component or any combination of variouscomponents, of the formulations of the invention is adjusted to achievethe desired tonicity of the final formulation. For example, the ratio ofthe carbohydrate excipient to antibody may be adjusted according tomethods known in the art (e.g., U.S. Pat. No. 6,685,940). In certainembodiments, the molar ratio of the carbohydrate excipient to antibodymay be from about 100 moles to about 1000 moles of carbohydrateexcipient to about 1 mole of antibody, or from about 200 moles to about6000 moles of carbohydrate excipient to about 1 mole of antibody, orfrom about 100 moles to about 510 moles of carbohydrate excipient toabout 1 mole of antibody, or from about 100 moles to about 600 moles ofcarbohydrate excipient to about 1 mole of antibody.

The desired isotonicity of the final formulation may also be achieved byadjusting the salt concentration of the formulations. Pharmaceuticallyacceptable salts and those suitable for this invention as tonicitymodifiers include, but are not limited to, sodium chloride, sodiumsuccinate, sodium sulfate, potassium chloride, magnesium chloride,magnesium sulfate, and calcium chloride. In specific embodiments,formulations of the invention comprise NaCl, MgCl₂, and/or CaCl₂. In oneembodiment, concentration of NaCl is between about 75 mM and about 150mM. In another embodiment, concentration of MgCl₂ is between about 1 mMand about 100 mM. Pharmaceutically acceptable amino acids includingthose suitable for this invention as tonicity modifiers include, but arenot limited to, proline, alanine, L-arginine, asparagine, L-asparticacid, glycine, serine, lysine, and histidine.

In one embodiment the formulations of the invention are pyrogen-freeformulations which are substantially free of endotoxins and/or relatedpyrogenic substances. Endotoxins include toxins that are confined insidea microorganism and are released only when the microorganisms are brokendown or die. Pyrogenic substances also include fever-inducing,thermostable substances (glycoproteins) from the outer membrane ofbacteria and other microorganisms. Both of these substances can causefever, hypotension and shock if administered to humans. Due to thepotential harmful effects, even low amounts of endotoxins must beremoved from intravenously administered pharmaceutical drug solutions.The Food & Drug Administration (“FDA”) has set an upper limit of 5endotoxin units (EU) per dose per kilogram body weight in a single onehour period for intravenous drug applications (The United StatesPharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). Whentherapeutic proteins are administered in amounts of several hundred orthousand milligrams per kilogram body weight, as can be the case withantibodies, even trace amounts of harmful and dangerous endotoxin mustbe removed. In certain specific embodiments, the endotoxin and pyrogenlevels in the composition are less then 10 EU/mg, or less then 5 EU/mg,or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg,or less then 0.001 EU/mg.

When used for in vivo administration, the formulations of the inventionshould be sterile. The formulations of the invention may be sterilizedby various sterilization methods, including sterile filtration,radiation, etc. In one embodiment, the antibody formulation isfilter-sterilized with a presterilized 0.22-micron filter. Sterilecompositions for injection can be formulated according to conventionalpharmaceutical practice as described in “Remington: The Science &Practice of Pharmacy”, 2^(st) ed., Lippincott Williams & Wilkins,(2005). Formulations comprising antibodies, such as those disclosedherein, ordinarily will be stored in lyophilized form or in solution. Itis contemplated that sterile compositions comprising antibodies areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having an adapter that allows retrievalof the formulation, such as a stopper pierceable by a hypodermicinjection needle. In one embodiment, a composition of the invention isprovided as a pre-filled syringe.

In one embodiment, a formulation of the invention is a lyophilizedformulation. The term “lyophilized” or “freeze-dried” includes a stateof a substance that has been subjected to a drying procedure such aslyophilization, where at least 50% of moisture has been removed.

The phrase “bulking agent” includes a compound that is pharmaceuticallyacceptable and that adds bulk to a lyo cake. Bulking agents known to theart include, for example, carbohydrates, including simple sugars such asdextrose, ribose, fructose and the like, alcohol sugars such asmannitol, inositol and sorbitol, disaccharides including trehalose,sucrose and lactose, naturally occurring polymers such as starch,dextrans, chitosan, hyaluronate, proteins (e.g., gelatin and serumalbumin), glycogen, and synthetic monomers and polymers.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest (such as an antibody of the invention), significantly preventsor reduces chemical and/or physical instability of the protein uponlyophilization and subsequent storage. Lyoprotectants include, but arenot limited to, sugars and their corresponding sugar alcohols; an aminoacid such as monosodium glutamate or histidine; a methylamine such asbetaine; a lyotropic salt such as magnesium sulfate; a polyol such astrihydric or higher molecular weight sugar alcohols, e.g., glycerin,dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, andmannitol; propylene glycol; polyethylene glycol; PLURONICS™; andcombinations thereof. Additional examples of lyoprotectants include, butare not limited to, glycerin and gelatin, and the sugars mellibiose,melezitose, raffinose, mannotriose and stachyose. Examples of reducingsugars include, but are not limited to, glucose, maltose, lactose,maltulose, iso-maltulose and lactulose. Examples of non-reducing sugarsinclude, but are not limited to, non-reducing glycosides of polyhydroxycompounds selected from sugar alcohols and other straight chainpolyalcohols. Examples of sugar alcohols include, but are not limitedto, monoglycosides, compounds obtained by reduction of disaccharidessuch as lactose, maltose, lactulose and maltulose. The glycosidic sidegroup can be either glucosidic or galactosidic. Additional examples ofsugar alcohols include, but are not limited to, glucitol, maltitol,lactitol and iso-maltulose. In specific embodiments, trehalose orsucrose is used as a lyoprotectant.

The lyoprotectant is added to the pre-lyophilized formulation in a“lyoprotecting amount” which means that, following lyophilization of theprotein in the presence of the lyoprotecting amount of thelyoprotectant, the protein essentially retains its physical and chemicalstability and integrity upon lyophilization and storage.

In one embodiment, the molar ratio of a lyoprotectant (e.g., trehalose)and antibody molecules of a formulation of the invention is at leastabout 10, at least about 50, at least about 100, at least about 200, orat least about 300. In another embodiment, the molar ratio of alyoprotectant (e.g., trehalose) and antibody molecules of a formulationof the invention is about 1, is about 2, is about 5, is about 10, about50, about 100, about 200, or about 300.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized antibody formulation in a diluent such that theantibody is dispersed in the reconstituted formulation. Thereconstituted formulation is suitable for administration (e.g.,parenteral administration) to a patient to be treated with the antibodyand, in certain embodiments of the invention, may be one which issuitable for intravenous administration.

The “diluent” of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation, such as aformulation reconstituted after lyophilization. In some embodiments,diluents include, but are not limited to, sterile water, bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution. In an alternative embodiment, diluents can includeaqueous solutions of salts and/or buffers.

In certain embodiments, a formulation of the invention is a lyophilizedformulation comprising an antibody of the invention, wherein at leastabout 90%, at least about 95%, at least about 97%, at least about 98%,or at least about 99% of said antibody may be recovered from a vial uponshaking said vial for 4 hours at a speed of 400 shakes per minutewherein the vial is filled to half of its volume with the formulation.In another embodiment, a formulation of the invention is a lyophilizedformulation comprising an antibody of the invention, wherein at leastabout 90%, at least about 95%, at least about 97%, at least about 98%,or at least about 99% of the antibody may be recovered from a vial uponsubjecting the formulation to three freeze/thaw cycles wherein the vialis filled to half of its volume with said formulation. In a furtherembodiment, a formulation of the invention is a lyophilized formulationcomprising an antibody of the invention, wherein at least about 90%, atleast about 95%, at least about 97%, at least about 98%, or at leastabout 99% of the antibody may be recovered by reconstituting alyophilized cake generated from said formulation.

In one embodiment, a reconstituted liquid formulation may comprise anantibody at the same concentration as the pre-lyophilized liquidformulation.

In another embodiment, a reconstituted liquid formulation may comprisean antibody at a higher concentration than the pre-lyophilized liquidformulation, e.g., about 2 fold, about 3 fold, about 4 fold, about 5fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about10 fold higher concentration of an antibody than the pre-lyophilizedliquid formulation.

In yet another embodiment, a reconstituted liquid formulation maycomprise an antibody of the invention at a lower concentration than thepre-lyophilized liquid formulation, e.g., about 2 fold, about 3 fold,about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold,about 9 fold or about 10 fold lower concentration of an antibody thanthe pre-lyophilized liquid formulation.

The pharmaceutical formulations of the invention are preferably stableformulations, e.g., stable at room temperature.

The terms “stability” and “stable” as used herein in the context of aformulation comprising an antibody of the invention refer to theresistance of the antibody in the formulation to aggregation,degradation or fragmentation under given manufacture, preparation,transportation and storage conditions. The “stable” formulations of theinvention retain biological activity under given manufacture,preparation, transportation and storage conditions. The stability of theantibody can be assessed by degrees of aggregation, degradation orfragmentation, as measured by HPSEC, static light scattering (SLS),Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD),urea unfolding techniques, intrinsic tryptophan fluorescence,differential scanning calorimetry, and/or ANS binding techniques,compared to a reference formulation. For example, a referenceformulation may be a reference standard frozen at −70° C. consisting of10 mg/ml of an antibody of the invention in PBS.

Therapeutic formulations of the present invention may be formulated fora particular dosage. Dosage regimens may be adjusted to provide theoptimum desired response (e.g., a therapeutic response). For example, asingle bolus may be administered, several divided doses may beadministered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the antibody and the particular therapeutic effect tobe achieved, and (b) the limitations inherent in the art of compoundingsuch an antibody for the treatment of sensitivity in individuals.

Therapeutic compositions of the present invention can be formulated forparticular routes of administration, such as oral, nasal, pulmonary,topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods knownin the art of pharmacy. The amount of active ingredient which can becombined with a carrier material to produce a single dosage form willvary depending upon the subject being treated, and the particular modeof administration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the composition which produces a therapeutic effect.By way of example, in certain embodiments, the antibodies (includingantibody fragments) are formulated for intravenous administration. Incertain other embodiments, the antibodies (including antibody fragments)are formulated for local delivery to the cardiovascular system, forexample, via catheter, stent, wire, intramyocardial delivery,intrapericardial delivery, or intraendocardial delivery.

Formulations of the present invention which are suitable for topical ortransdermal administration include powders, sprays, ointments, pastes,creams, lotions, gels, solutions, patches and inhalants. The activecompound may be mixed under sterile conditions with a pharmaceuticallyacceptable carrier, and with any preservatives, buffers, or propellantswhich may be required (U.S. Pat. Nos. 7,378,110; 7,258,873; 7,135,180;US Publication No. 2004-0042972; and 2004-0042971).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

In certain embodiments, antibodies of the invention can be formulated toensure proper distribution in vivo. For example, the blood-brain bather(BBB) excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds of the invention can cross the BBB (if desired),they can be formulated, for example, in liposomes. For methods ofmanufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548;5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin.Pharmacol. 29:685). Exemplary targeting moieties include folate orbiotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al.,(1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G.Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995)Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor(Briscoe et al. (1995) Am. J. Physiol. 1233:134), different species ofwhich may comprise the formulations of the invention, as well ascomponents of the invented molecules; p120 (Schreier et al. (1994) J.Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBSLett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.In one embodiment of the invention, the therapeutic compounds of theinvention are formulated in liposomes; in another embodiment, theliposomes include a targeting moiety. In another embodiment, thetherapeutic compounds in the liposomes are delivered by bolus injectionto a site proximal to the desired area. When administered in thismanner, the composition must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and may be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. Additionally oralternatively, the antibodies of the invention may be delivered locallyto the brain to mitigate the risk that the blood brain barrier slowseffective delivery.

In certain embodiments, the therapeutic antibody compositions may beadministered with medical devices known in the art. For example, incertain embodiments an antibody or antibody fragment is administeredlocally via a catheter, stent, wire, or the like. For example, in oneembodiment, a therapeutic composition of the invention can beadministered with a needleless hypodermic injection device, such as thedevices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824; 4,596,556. Examples of well-knownimplants and modules useful in the present invention include: U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicants throughthe skin; U.S. Pat. No. 4,447,233, which discloses a medication infusionpump for delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. Many other such implants, delivery systems, andmodules are known to those skilled in the art.

The efficient dosages and the dosage regimens for the antibodies of theinvention depend on the disease or condition to be treated and can bedetermined by the persons skilled in the art. One of ordinary skill inthe art would be able to determine such amounts based on such factors asthe subject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

a. Alternative Aqueous Formulations

The invention also provides compositions comprising an antibody, orantigen-binding portion thereof, comprising a PGPK modificationformulated as an aqueous formulation comprising a protein and water, asdescribed in U.S. Pat. No. 8,420,081 and PCT Publication No.WO2012/065072, the contents of each of which are hereby incorporated byreference. In these aqueous formulations, the antibody, orantigen-binding portion thereof, comprising a PGPK modification isstable without the need for additional agents. This aqueous formulationhas a number of advantages over conventional formulations in the art,including stability of the protein in water without the requirement foradditional excipients, increased concentrations of protein without theneed for additional excipients to maintain solubility of the protein,and low osmolality. These also have advantageous storage properties, asthe proteins in the formulation remain stable during storage, e.g.,stored as a liquid form for more than 3 months at 7° C. or freeze/thawconditions, even at high protein concentrations and repeated freeze/thawprocessing steps. In one embodiment, formulations described hereininclude high concentrations of antibodies, or antigen-binding portionsthereof, comprising a PGPK modification such that the aqueousformulation does not show significant opalescence, aggregation, orprecipitation.

In one embodiment, a composition comprising an antibody, orantigen-binding portion thereof, comprising a PGPK modification andwater is provided, wherein the formulation has certain characteristics,such as, but not limited to, low conductivity, e.g., a conductivity ofless than about 2.5 mS/cm, a protein concentration of at least about 10μg/mL, an osmolality of no more than about 30 mOsmol/kg, and/or theprotein has a molecular weight (Mw) greater than about 47 kDa. In oneembodiment, the formulation has improved stability, such as, but notlimited to, stability in a liquid form for an extended time (e.g., atleast about 3 months or at least about 12 months) or stability throughat least one freeze/thaw cycle (if not more freeze/thaw cycles). In oneembodiment, the formulation is stable for at least about 3 months in aform selected from the group consisting of frozen, lyophilized, orspray-dried.

In one embodiment, the formulation has a low conductivity, including,for example, a conductivity of less than about 2.5 mS/cm, a conductivityof less than about 2 mS/cm, a conductivity of less than about 1.5 mS/cm,a conductivity of less than about 1 mS/cm, or a conductivity of lessthan about 0.5 mS/cm.

In another embodiment, antibodies, or antigen-binding portions thereof,comprising a PGPK modification of the invention included in theformulation have a given concentration, including, for example, aconcentration of at least about 1 mg/mL, at least about 10 mg/mL, atleast about 50 mg/mL, at least about 100 mg/mL, at least about 150mg/mL, at least about 200 mg/mL, or greater than about 200 mg/mL. Inanother embodiment, the formulation of the invention has an osmolalityof no more than about 15 mOsmol/kg.

The aqueous formulations described herein do not rely on standardexcipients, e.g., a tonicity modifier, a stabilizing agent, asurfactant, an anti-oxidant, a cryoprotectant, a bulking agent, alyroprotectant, a basic component, and an acidic component. In otherembodiments of the invention, the formulation contains water, one ormore proteins, and no ionic excipients (e.g., salts, free amino acids).

In certain embodiments, the aqueous formulations as described hereincomprise a composition comprising an antibody, or antigen-bindingportion thereof, comprising a PGPK modification having a proteinconcentration of at least 50 mg/mL and water, wherein the formulationhas an osmolality of no more than 30 mOsmol/kg. Lower limits ofosmolality of the aqueous formulation are also encompassed by theinvention. In one embodiment the osmolality of the aqueous formulationis no more than 15 mOsmol/kg. The aqueous formulation of the inventionmay have an osmolality of less than 30 mOsmol/kg, and also have a highprotein concentration, e.g., the concentration of the protein is atleast 100 mg/mL, and may be as much as 200 mg/mL or greater. Rangesintermediate to the above recited concentrations and osmolality unitsare also intended to be part of this invention. In addition, ranges ofvalues using a combination of any of the above recited values as upperand/or lower limits are intended to be included.

The concentration of the aqueous formulation as described herein is notlimited by the protein size and the formulation may include any sizerange of proteins. Included within the scope of the invention is anaqueous formulation comprising at least 40 mg/mL and as much as 200mg/mL or more of a protein, for example, 40 mg/mL, 65 mg/mL, 130 mg/mL,or 195 mg/ml, which may range in size from 5 kDa to 150 kDa or more. Inone embodiment, the protein in the formulation of the invention is atleast about 15 kD in size, at least about 20 kD in size; at least about47 kD in size; at least about 60 kD in size; at least about 80 kD insize; at least about 100 kD in size; at least about 120 kD in size; atleast about 140 kD in size; at least about 160 kD in size; or greaterthan about 160 kD in size. Ranges intermediate to the above recitedsizes are also intended to be part of this invention. In addition,ranges of values using a combination of any of the above recited valuesas upper and/or lower limits are intended to be included.

The aqueous formulation as described herein may be characterized by thehydrodynamic diameter (D_(h)) of the proteins in solution. Thehydrodynamic diameter of the protein in solution may be measured usingdynamic light scattering (DLS), which is an established analyticalmethod for determining the D_(h) of proteins. Typical values formonoclonal antibodies, e.g., IgG, are about 10 nm Low-ionic formulationsmay be characterized in that the D_(h) of the proteins are notably lowerthan protein formulations comprising ionic excipients. It has beendiscovered that the D_(h) values of antibodies in aqueous formulationsmade using the disfiltration/ultrafilteration (DF/UF) process, asdescribed in U.S. Pat. No. 8,420,081 and PCT Publication No.WO2012/065072, using pure water as an exchange medium, are notably lowerthan the D_(h) of antibodies in conventional formulations independent ofprotein concentration. In one embodiment, antibodies in the aqueousformulation as described herein have a D_(h) of less than 4 nm, or lessthan 3 nm.

In one embodiment, the D_(h) of the protein in the aqueous formulationis smaller relative to the D_(h) of the same protein in a bufferedsolution, irrespective of protein concentration. Thus, in certainembodiments, protein in an aqueous formulation made in accordance withthe methods described herein, will have a D_(h) which is at least 25%less than the D_(h) of the protein in a buffered solution at the samegiven concentration. Examples of buffered solutions include, but are notlimited to phosphate buffered saline (PBS). In certain embodiments,proteins in the aqueous formulation of the invention have a D_(h) thatis at least 50% less than the D_(h) of the protein in PBS in at thegiven concentration; at least 60% less than the D_(h) of the protein inPBS at the given concentration; at least 70% less than the D_(h) of theprotein in PBS at the given concentration; or more than 70% less thanthe D_(h) of the protein in PBS at the given concentration. Rangesintermediate to the above recited percentages are also intended to bepart of this invention, e.g., 55%, 56%, 57%, 64%, 68%, and so forth. Inaddition, ranges of values using a combination of any of the aboverecited values as upper and/or lower limits are intended to be included,e.g., 50% to 80%.

In one aspect, the aqueous formulation includes the antibody, orantigen-binding portion thereof, comprising a PGPK modification at adosage of about 0.01 mg/kg-10 mg/kg. In another aspect, the dosages ofthe antibody, or antigen-binding portion thereof, comprising a PGPKmodification include approximately 1 mg/kg administered every otherweek, or approximately 0.3 mg/kg administered weekly. A skilledpractitioner can ascertain the proper dosage and regime foradministering to a subject.

b. “Solid Unit” Formulations

The present invention also provides stable solid compositions of aprotein (preferably a therapeutic protein) and a stabilizer, referred toherein as solid units. These formulations are described, for example, inU.S. Provisional Patent Application 61/893,123, entitled “Stable SolidProtein Compositions and Methods of Making Same”, filed on Oct. 18,2013, the entire contents of which are expressly incorporated herein byreference. Specifically, it has been discovered that despite having ahigh proportion of sugar relative to the protein, the solid units of theinvention maintain structural rigidity and resist changes in shapeand/or volume when stored under ambient conditions, e.g., roomtemperature and humidity, for extended periods of time. The solid unitsof the invention remain free-flowing and are able to maintain long-termphysical and chemical stability of the protein without significantdegradation and/or aggregate formation. The solid units of the inventionhave many advantages over the art, including that they can be formulatedfor oral delivery and are easily reconstituted in a diluent, such aswater. Because the solid units are readily dissolved, they may be usedin dual chamber delivery devices and may be prepared directly in adevice for patient use.

As used herein, the term “solid unit,” refers to a composition which issuitable for pharmaceutical administration and comprises a protein,e.g., an antibody or peptide, and a stabilizer, e.g., a sugar. The solidunit has a structural rigidity and resistance to changes in shape and/orvolume. In a preferred embodiment, the solid unit is obtained bylyophilizing a pharmaceutical formulation of a therapeutic protein. Thesolid unit may be any shape, e.g., geometric shape, including, but notlimited to, a sphere, a cube, a pyramid, a hemisphere, a cylinder, ateardrop, and so forth, including irregularly shaped units. In oneembodiment, the solid unit has a volume ranging from about 1 μl to about204 μl. In one embodiment, the solid unit is not obtained using spraydrying techniques, e.g., the solid unit is not a powder or granule.

As used herein, the phrase “a plurality of solid units” refers to acollection or population of solid units, wherein the collectioncomprises two or more solid units having a substantially uniform shape,e.g., sphere, and/or volume distribution. In one embodiment, theplurality of solid units is free-flowing.

VI. Kits and Articles of Manufacture Comprising Antibodies of theInvention

Also within the scope of the present invention are kits comprising theantibodies, and antigen-binding portions thereof, of the invention andinstructions for use. The term “kit” as used herein refers to a packagedproduct comprising components with which to administer the antibody, orantigen-binding portion thereof, of the invention for treatment of adisease or disorder. The kit typically comprises a box or container thatholds the components of the kit. The box or container is affixed with alabel or a Food and Drug Administration approved protocol. The box orcontainer holds components of the invention which are typicallycontained within plastic, polyethylene, polypropylene, ethylene, orpropylene vessels. The vessels can be capped-tubes or bottles. The kitcan also include instructions for administering an antibody of theinvention.

The kit can further contain one more additional reagents, such as animmunosuppressive reagent, a cytotoxic agent or a radiotoxic agent orone or more additional antibodies of the invention (e.g., an antibodyhaving a complementary activity which binds to an epitope in the TNFαantigen distinct from a first anti-TNFα antibody). Kits typicallyinclude a label indicating the intended use of the contents of the kit.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with a liquid formulation or lyophilizedformulation of an antibody or antibody fragment thereof of theinvention. In one embodiment, a container filled with a liquidformulation of the invention is a pre-filled syringe. In a specificembodiment, the formulations of the invention are formulated in singledose vials as a sterile liquid. For example, the formulations may besupplied in 3 cc USP Type I borosilicate amber vials (WestPharmaceutical Services—Part No. 6800-0675) with a target volume of 1.2mL. Optionally associated with such container(s) can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

In one embodiment, a container filled with a liquid formulation of theinvention is a pre-filled syringe. Any pre-filled syringe known to oneof skill in the art may be used in combination with a liquid formulationof the invention. Pre-filled syringes that may be used are described in,for example, but not limited to, PCT Publications WO05032627,WO08094984, WO9945985, WO03077976, U.S. Pat. Nos. 6,792,743, 5,607,400,5,893,842, 7,081,107, 7,041,087, 5,989,227, 6,807,797, 6,142,976,5,899,889, US Patent Publications US20070161961A1, US20050075611A1,US20070092487A1, US20040267194A1, US20060129108A1. Pre-filled syringesmay be made of various materials. In one embodiment a pre-filled syringeis a glass syringe. In another embodiment a pre-filled syringe is aplastic syringe. One of skill in the art understands that the natureand/or quality of the materials used for manufacturing the syringe mayinfluence the stability of a protein formulation stored in the syringe.For example, it is understood that silicon based lubricants deposited onthe inside surface of the syringe chamber may affect particle formationin the protein formulation. In one embodiment, a pre-filled syringecomprises a silicone based lubricant. In one embodiment, a pre-filledsyringe comprises baked on silicone. In another embodiment, a pre-filledsyringe is free from silicone based lubricants. One of skill in the artalso understands that small amounts of contaminating elements leachinginto the formulation from the syringe barrel, syringe tip cap, plungeror stopper may also influence stability of the formulation. For example,it is understood that tungsten introduced during the manufacturingprocess may adversely affect formulation stability. In one embodiment, apre-filled syringe may comprise tungsten at a level above 500 ppb. Inanother embodiment, a pre-filled syringe is a low tungsten syringe. Inanother embodiment, a pre-filled syringe may comprise tungsten at alevel between about 500 ppb and about 10 ppb, between about 400 ppb andabout 10 ppb, between about 300 ppb and about 10 ppb, between about 200ppb and about 10 ppb, between about 100 ppb and about 10 ppb, betweenabout 50 ppb and about 10 ppb, between about 25 ppb and about 10 ppb.

In certain embodiments, kits comprising antibodies of the invention arealso provided that are useful for various purposes, e.g., research anddiagnostic including for purification or immunoprecipitation of proteinof interest from cells, detection of the protein of interest in vitro orin vivo. For isolation and purification of a protein of interest, thekit may contain an antibody coupled to beads (e.g., sepharose beads).Kits may be provided which contain the antibodies for detection andquantitation of a protein of interest in vitro, e.g., in an ELISA or aWestern blot. As with the article of manufacture, the kit comprises acontainer and a label or package insert on or associated with thecontainer. The container holds a composition comprising at least oneantibody of the invention. Additional containers may be included thatcontain, e.g., diluents and buffers, control antibodies. The label orpackage insert may provide a description of the composition as well asinstructions for the intended in vitro or diagnostic use.

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial, pre-filled syringe or other container that ishermetically sealed. In one embodiment, the unit dosage form is providedas a sterile particulate free solution comprising an antibody that issuitable for parenteral administration. In another embodiment, the unitdosage form is provided as a sterile lyophilized powder comprising anantibody that is suitable for reconstitution.

In one embodiment, the unit dosage form is suitable for intravenous,intramuscular, intranasal, oral, topical or subcutaneous delivery. Thus,the invention encompasses sterile solutions suitable for each deliveryroute. The invention further encompasses sterile lyophilized powdersthat are suitable for reconstitution.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question, as well as how and how frequently toadminister the pharmaceutical. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, and other monitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, pre-filled syringe, sprayer, insufflator, intravenous (i.v.)bag, envelope and the like; and at least one unit dosage form of apharmaceutical agent contained within said packaging material, whereinsaid pharmaceutical agent comprises a liquid formulation containing anantibody. The packaging material includes instruction means whichindicate how that said antibody can be used to prevent, treat and/ormanage one or more symptoms associated with a disease or disorder.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references, including literature references, issued patents,and published patent applications, as cited throughout this applicationare hereby expressly incorporated herein by reference. It should furtherbe understood that the contents of all the figures and tables attachedhereto are expressly incorporated herein by reference. The entirecontents of the following applications are also expressly incorporatedherein by reference: U.S. Provisional Patent Application 61/893,123,entitled “STABLE SOLID PROTEIN COMPOSITIONS AND METHODS OF MAKING SAME”,filed on Oct. 18, 2013; U.S. Provisional Application Ser. No.61/892,833, entitled “LOW ACIDIC SPECIES COMPOSITIONS AND METHODS FORPRODUCING THE SAME USING DISPLACEMENT CHROMATOGRAPHY”, filed on Oct. 18,2013; U.S. Provisional Patent Application 61/892,710, entitled “MUTATEDANTI-TNFα ANTIBODIES AND METHODS OF THEIR USE”, filed on Oct. 18, 2013;U.S. Provisional Patent Application 61/893,068, entitled “LOW ACIDICSPECIES COMPOSITIONS AND METHODS FOR PRODUCING THE SAME”, filed on Oct.18, 2013; U.S. Provisional Patent Application 61/893,088, entitled“MODULATED LYSINE VARIANT SPECIES AND METHODS FOR PRODUCING AND USINGTHE SAME”, filed on Oct. 18, 2013; and U.S. Provisional PatentApplication 61/893,131, entitled “PURIFICATION OF PROTEINS USINGHYDROPHOBIC INTERACTION CHROMATOGRAPHY”, filed on Oct. 18, 2013.

EXAMPLES Example 1 Analysis of Product-Related Substance Variation ofAdalimumab

The production of proteins for biopharmaceutical applications typicallyinvolves the use of cell cultures that are known to produce proteinsexhibiting varying levels of product-related substance heterogeneity.Such heterogeneity includes, but is not limited to, charge variants suchas acidic species and basic species. For example, but not by way oflimitation, the charge variants can be separated based onchromatographic residence time. FIG. 1 depicts a non-limiting example ofsuch a division wherein the total acidic species associated with theexpression of Adalimumab is divided into a first acidic species region(AR1) and a second acidic species region (AR2), as well as the unchargedproduct (Lys 0) and the two basic variants, where one C-terminal lysineis present (Lys 1) or both C-terminal lysines are present (Lys 2). Thecompositions of particular acidic species regions may differ dependingon the particular antibody of interest, as well as the particular cellculture, purification, and/or chromatographic conditions employed. Asdepicted in FIG. 2, the individual charge variants can be resolved fromeach other.

Example 2 Reduction in Lysine Variation by Treatment withCarboxypeptidase B

Without being bound by theory, it is believed that a C-terminal lysineof antibody will be quickly cleaved enzymatically through the catalysisof carboxypeptidase U. In order to test the susceptibility of antibodiesto such enzymatic removal, which would convert Lys 1 and Lys 2 speciesto Lys 0, a recombinant carboxypeptidase B was incubated with anAdalimumab sample containing variants, including those with terminallysines. The population of antibody with C-terminal lysine was quicklyconverted to Lys 0 species as shown in FIG. 3 (compare untreated whichincludes a population of Lys 1 to CPB treated where essentially theentire population has been converted to Lys 0).

Example 3 Preparation of Modified Anti-TNFα Antibody

In order to minimize enzymatic cleavage of the C-terminal lysine, andthereby reduce product heterogeneity, a modified anti-TNFα antibody wasprepared. Specifically, a proline residue was inserted between theC-terminal lysine and the immediately preceding glycine. Thus theC-terminal three amino acids were altered from the original AdalimumabC-terminal sequence, PGK, to a modified C-terminal sequence of PGPK.Without being bound by theory, it is believed that because proline is animino acid in which the side chain bonds to its backbone nitrogen, theinclusion of a prolines residue at this location will add a kink andrigidity to the peptide backbone and as such will restrict the abilityof peptidases to cleave the C-terminal lysine.

Example 4 Physical Properties of Modified Anti-TNFα Antibody

In order to compare the physical properties the modified anti-TNFαantibody prepared above using Adalimumab, the following series ofexperiments were performed.

Adalimumab and the modified anti-TNFα antibody were incubated in humanand rat plasma in order to compare their susceptibility to C-terminalcleavage by plasma proteases. The carboxypeptidase U found in serumcoordinates a divalent metal cation in its catalysis. Citrate is aneffective anticoagulant but is also a chelator which may strip thecation from the active site and inhibit the enzyme's activity. Toaddress these possible issues, the instant experiment investigatesC-terminal processing of either Adalimumab or the modified anti-TNFαantibody in the presence of human plasma with citrate as ananticoagulant, human plasma with heparin as an anticoagulant and mouseplasma with heparin as an anticoagulant. The data is presented in FIG. 6(Adalimumab) and 7 (the modified anti-TNFα antibody). The modifiedanti-TNFα antibody was resistant to C-terminal processing when spikedinto each of the three different plasma samples. In contrast,Adalimumab, including a sub-population of antibody with C-terminallysine, was only resistant to processing in the plasma sample thatincluded citrate as an anticoagulant. These data indicate that thechelation of the citrate to the carboxypeptidase U active site inhibitsthe processing. The two plasma samples with heparin as an anticoagulanthave no residual C-terminal lysine constituents. In summary, the datademonstrates that the endogenous carboxypeptidase are capable ofprocessing the normal C-terminus of an antibody but cannot process themodified C-terminus thus allowing it to retain a localized net positivecharge.

In a second experiment, mice were immunized at 2 mg/Kg and 5 mg/Kg ofthe modified anti-TNFα antibody. The mice were then sacrificed and theterminal bleeds were obtained. The samples were passed through animmobilized TNF-alpha column and the eluate was analyzed by LC/MS toevaluate the presence or absence of an intact C-terminal lysine. Asdepicted in FIG. 7, the modified anti-TNFα antibody retained bothC-terminal lysine residues present when analyzed by LC/MS, thus themodified anti-TNFα antibody is able to retain the C-terminal lysines invivo.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. An antibody, or antigen-binding portionthereof, comprising a C-terminal heavy chain sequence ofproline-glycine-proline-lysine (PGPK) (SEQ ID NO:9).
 2. The antibody, orantigen-binding portion thereof, of claim 1, wherein the antibody, orantigen-binding portion thereof, is an anti-TNFα antibody, orantigen-binding portion thereof.
 3. A method of treating a subjecthaving a disorder in which TNFα activity is detrimental, the methodcomprising administering a therapeutically effective amount of theantibody, or antigen-binding portion thereof, of claim 2 to the subject,thereby treating the TNFα-associated disease or disorder.
 4. The methodof claim 3, wherein the disorder in which TNFα activity is detrimentalis selected from the group consisting of rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Crohn's Disease, plaque psoriasis,active axial spondyloarthritis and non-radiographic axialspondyloarthritis.
 5. The antibody, or antigen-binding portion thereof,of claim 2, wherein the antibody, or antigen-binding portion thereof,comprises the heavy and light chain variable regions of adalimumab.
 6. Apharmaceutical composition comprising the antibody, or antigen-bindingportion thereof, of claim 5 and a pharmaceutically acceptable carrier.7. A pharmaceutical composition comprising the antibody, orantigen-binding portion thereof, of claim 2 and a pharmaceuticallyacceptable carrier.
 8. A pharmaceutical composition comprising theantibody, or antigen-binding portion thereof, of claim 1 and apharmaceutically acceptable carrier.
 9. An anti-TNFα antibody, orantigen-binding portion thereof, comprising a C-terminal heavy chainsequence of proline-glycine-proline-lysine (PGPK) (SEQ ID NO:9).
 10. Apharmaceutical composition comprising the antibody, or antigen-bindingportion thereof, of claim 9 and a pharmaceutically acceptable carrier.11. A method of treating a subject having a disorder in which TNFαactivity is detrimental, the method comprising administering atherapeutically effective amount of the antibody, or antigen-bindingportion thereof, of claim 9 to the subject, thereby treating theTNFα-associated disease or disorder.
 12. The method of claim 11, whereinthe disorder in which TNFα activity is detrimental is selected from thegroup consisting of rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Crohn's Disease, plaque psoriasis, active axialspondyloarthritis and non-radiographic axial spondyloarthritis.
 13. Anantibody, or antigen-binding portion thereof, comprising the heavy andlight chain variable regions of adalimumab and a C-terminal heavy chainsequence of proline-glycine-proline-lysine (PGPK) (SEQ ID NO:9).
 14. Apharmaceutical composition comprising the antibody, or antigen-bindingportion thereof, of claim 13 and a pharmaceutically acceptable carrier.15. A method of treating a subject having a disorder in which TNFαactivity is detrimental, the method comprising administering atherapeutically effective amount of the antibody, or antigen-bindingportion thereof, of claim 13 to the subject, thereby treating theTNFα-associated disease or disorder.
 16. The method of claim 15, whereinthe disorder in which TNFα activity is detrimental is selected from thegroup consisting of rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Crohn's Disease, plaque psoriasis, active axialspondyloarthritis and non-radiographic axial spondyloarthritis.
 17. Apharmaceutical composition comprising an antibody, or antigen-bindingportion thereof, comprising a C-terminal heavy chain sequence ofproline-glycine-proline-lysine (PGPK) (SEQ ID NO:9) and apharmaceutically acceptable carrier.
 18. A method of treating a subjecthaving a disorder in which TNFα activity is detrimental, the methodcomprising administering a therapeutically effective amount of anantibody, or antigen-binding portion thereof, comprising the heavy andlight chain variable regions of adalimumab and a C-terminal heavy chainsequence of proline-glycine-proline-lysine (PGPK) (SEQ ID NO:9) to thesubject, thereby treating the TNFα-associated disease or disorder. 19.The method of claim 18, wherein the disorder in which TNFα activity isdetrimental is selected from the group consisting of rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's Disease,plaque psoriasis, active axial spondyloarthritis and non-radiographicaxial spondyloarthritis.
 20. A pharmaceutical composition comprising ananti-TNFα antibody, or antigen-binding portion thereof, comprising aC-terminal heavy chain sequence of proline-glycine-proline-lysine (PGPK)(SEQ ID NO:9) and a pharmaceutically acceptable carrier.
 21. A method oftreating a subject having a disorder in which TNFα activity isdetrimental, the method comprising administering the pharmaceuticalcomposition of claim 20 to the subject, thereby treating theTNFα-associated disease or disorder.
 22. The method of claim 21, whereinthe disorder in which TNFα activity is detrimental is selected from thegroup consisting of rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Crohn's Disease, plaque psoriasis, active axialspondyloarthritis and non-radiographic axial spondyloarthritis.
 23. Apharmaceutical composition comprising an antibody, or antigen-bindingportion thereof, comprising the heavy and light chain variable regionsof adalimumab and a C-terminal heavy chain sequence ofproline-glycine-proline-lysine (PGPK) (SEQ ID NO:9), and apharmaceutically acceptable carrier.
 24. A method of treating a subjecthaving a disorder in which TNFα activity is detrimental, the methodcomprising administering the pharmaceutical composition of claim 23 tothe subject, thereby treating the TNFα-associated disease or disorder.25. The method of claim 24, wherein the disorder in which TNFα activityis detrimental is selected from the group consisting of rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's Disease,plaque psoriasis, active axial spondyloarthritis and non-radiographicaxial spondyloarthritis.